ST2 antigen binding proteins

ABSTRACT

Described herein are compositions and methods related to antigen binding proteins that bind to human ST2, including antibodies. In particular embodiments, the disclosure provides fully human anti-ST2 antibodies and deriviatives and variants thereof. Further provided are nucleic acids encoding such antibodies and antibody fragments, variants, and derivatives. Also, provided are methods of making and using such antibodies including methods of treating and preventing autoimmune and inflammatory disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/792,619, filed Mar. 15, 2013, and U.S. Provisional PatentApplication No. 61/649,147, filed May 18, 2012, which are incorporatedherein by reference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format via EFS-Web. The Sequence Listing is provided as atext file entitled A1712USNP_ST25.txt, created May 17, 2013, which is189,576 bytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

ST2 is a binding receptor for interleukin-33 (IL-33), a cytokine relatedto IL-1 and IL-18 and also known as NF-HEV or IL-1F11. ST2 is expressedas both a soluble non-signaling variant (soluble ST2 or sST2) and afull-length membrane-spanning form (FL ST2, ST2 or ST2L) that mediatescellular responses to IL-33. The latter form is expressed on a widerange of cell types implicated in pathologic inflammation in a number ofdisease settings. These include lymphocytes, particularly IL-5 andIL-13-expressing T helper cells, natural killer (NK) and naturalkiller-T (NKT) cells, as well as many so-called innate immune cells,such as mast cells, basophils, eosinophils, macrophages and innatehelper cells (also known as nuocytes (Neill, Wong et al. 2010)). IL-33binding to ST2 on these cells leads to the recruitment of abroadly-expressed co-receptor known as the IL-R Accessory Protein (AcP)and the activation of pro-inflammatory signaling, similar to IL-1 andIL-18. IL-33 is thus able to directly activate ST2-expressing cells orenhance their activation when in the presence of other activatingstimuli. Examples of IL-33-induced cellular responses include theproduction of inflammatory cytokines, such as IL-5, IL-6, IL-13, TNF,IFN-γ and GM-CSF as well as the production of chemokines, such as CXCL8,CCL17 and CCL24. IL-33 has also been shown to enhance acute allergicresponses by augmenting mast cell and basophil activation triggered byIgE receptor signaling or other mast cell and basophil activators. IL-33will also enhance the recruitment, survival and adhesive properties ofST2 expressing immune cells and thus is important in provoking andsustaining cellular inflammation in local tissues.

The pro-inflammatory actions of IL-33 on innate and adaptive immunecells culminate to promote a number of pathologic processes. In thelungs, these include increased airway inflammation, mucus production,airway hyper responsiveness and fibrotic remodeling. IL-33 can alsocontribute to localized inflammation in the joints as well as cutaneousand articular hypernociception, by promoting the production ofproinflammatory cytokines (Verri, Guerrero et al. 2008; Xu, Jiang et al.2008). Excessive IL-33 has been linked to pathologic collagen depositionand fibrosis and also contributes to epithelial damage in the setting ofinflammatory bowel disease. Through its potent effects on basophils andIgE-sensitized mast cells, IL-33 can also trigger anaphylactic shock(Pushparaj, Tay et al. 2009) and may play a contributing role inallergic disease. Many of these diseases are chronic and progressive innature and difficult to treat and there is a need for more effectivetreatments.

Consistent with its documented biologic effects, there are several linesof evidence that the IL-33/ST2 pathway contributes to human disease. Forexample, abnormally high expression of IL-33 is found in diseasesinvolving inflammation in mucosal tissues and articular inflammation.These include asthma (Prefontaine, Lajoie-Kadoch et al. 2009;Prefontaine, Nadigel et al. 2010), inflammatory bowel disease (Beltran,Nunez et al. 2010; Pastorelli, Garg et al. 2010; Sponheim, Pollheimer etal. 2010) and rheumatoid arthritis (Palmer, Talabot-Aver et al. 2009;Matsuyama, Okazaki et al. 2010). IL-33 expression is elevated inpsoriatic skin (Theoharides, Zhang et al. 2010) and the skin of atopicdermatitis patients (Pushparaj, Tay et al. 2009) and is also increasedin pathologic settings of fibrosis, such as systemic sclerosis (Yanaba.Yoshizaki et al. 2011) (Manetti, Ibba-Manneschi et al. 2009) and liverfibrosis (Marvie, Lisbonne et al. 2009). The concentration ofcirculating soluble ST2 is also elevated in numerous disease situations,further indicating a link between this cytokine pathway and thesediseases. Examples include asthma (Kuroiwa, Arai et al. 2001; Oshikawa.Kuroiwa et al. 2001; Ali, Zhang et al. 2009), chronic obstructivepulmonary disease (Hacker, Lambers et al. 2009), pulmonary fibrosis(Tajima, Oshikawa et al. 2003), sepsis and trauma (Brunner. Krenn et al.2004), HIV infection (Miyagaki, Sugaya et al. 2011), systemic lupuserythematosus (Mok, Huang et al. 2010), inflammatory bowel disease(Beltran, Nunez et al. 2010) as well as rheumatoid arthritis, sclerosis,Wegener's granulomatosis and Behchet disease (Kuroiwa, Arai et al. 2001)and cardiovascular disease (Shah and Januzzi 2010). IL-33 potentiateseosinophilic inflammation and there is evidence this pathway is involvedin eosinophil-associated disease, such as rhinosinusitis and nasalpolyposis (Plager, Kahl et al. 2010) and eosinophilic bronchitis(Oshikawa, Kuroiwa et al. 2001).

Additional evidence linking the IL-33/ST2 pathway to human disease isprovided by genetic studies, which have identified IL-33 and/or ST2 genepolymorphisms in the general population that are significantlyassociated with increased risk of disease or parameters of diseaseseverity. Several large genome-wide association studies have linkedgenetic variation in ST2 (IL1RL1) or IL-33 with increased risk of asthma(Gudbjartsson, Bjornsdottir et al. 2009; Moffatt, Gut et al. 2010; Wu,Romieu et al. 2010) and other studies have genetically linked thispathway to increased asthma severity (Ali, Zhang et al. 2009) andbronchial hyper responsiveness (Reijmerink, Postma et al. 2008). Similarfindings have genetically implicated this pathway in allergic disorderssuch as atopic dermatitis (Shimizu, Matsuda et al. 2005), rhinosinusitis(Sakashita, Yoshimoto et al. 2008; Castano R 2009) as well as nasalpolyposis (Buysschaert, Grulois et al. 2010).

Collectively, these links to several human diseases and the ability ofthis cytokine axis to promote many forms of harmful inflammation implythis is a useful target for therapeutic intervention.

SUMMARY OF THE INVENTION

The invention provides anti-ST2 antigen binding proteins, e.g.,antibodies and functional fragments thereof, having properties amenableto commercial production and therapeutic use in humans. The anti-ST2antigen binding proteins are particularly useful in methods of treatingdiseases and disorders associated with the IL-33/ST2 axis. Providedherein are ST2-binding antibodies that bind ST2 with high affinity andeffectively block IL-33-binding, thereby reducing IL-33-mediatedsignaling in the cell.

In a first aspect, the ST2 antigen binding protein comprises a) a lightchain variable domain having at least 90% identity, at least 95%identity, or is identical to the amino acid sequence set forth in SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQID NO:105, SEQ ID NO:163, SEQ ID NO:164, or SEQ ID NO:165; b) a heavychain variable domain having at least 90% identity, at least 95%identity, or is identical to the amino acid sequence set forth in SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:145, SEQ ID NO:146, or SEQ ID NO:147; or c) the lightchain variable domain of a) and the heavy chain variable domain of b).

Preferred antigen binding proteins of the first aspect include thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:95 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:29; those comprising a light chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:96 and a heavy chain variable domain having at least 90%,at least 95%, or is identical to the amino acid sequence set forth inSEQ ID NO:30; those comprising a light chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:97 and a heavy chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:31; those comprising a light chain variable domain havingat least 90%, at least 95%, or is identical to the amino acid sequenceset forth in SEQ ID NO:98 and a heavy chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:32; those comprising a light chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:99 and a heavy chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:33; those comprising a light chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:100 and a heavy chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:34; those comprising alight chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:101 and aheavy chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:35; thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:102 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:36; those comprising a light chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:103 and a heavy chain variable domain having at least 90%,at least 95%, or is identical to the amino acid sequence set forth inSEQ ID NO:37; those comprising a light chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:104 and a heavy chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:38; those comprising a light chain variable domain havingat least 90%, at least 95%, or is identical to the amino acid sequenceset forth in SEQ ID NO:105 and a heavy chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:39; those comprising a light chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:163 and a heavy chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:145; those comprising a light chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:164 and a heavy chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:146; and those comprisinga light chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:165 and aheavy chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:147.

In a second aspect, the ST2 antigen binding protein comprises a) a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103,SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:163, SEQ ID NO:164, or SEQ IDNO:165; b) a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:145, SEQ IDNO:146, or SEQ ID NO:147; or c) the light chain variable domain of a)and the heavy chain variable domain of b).

Preferred antigen binding proteins of the second aspect include thosecomprising a light chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:95 and a heavy chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:29; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:96 and a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:30; those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:97 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:31;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:98 and a heavy chainvariable domain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:32; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:99 and a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:33; those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:100 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:34;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:101 and a heavychain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:35; those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:102 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:36;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:103 and a heavychain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:37; those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:104 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:38;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:105 and a heavychain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:39; those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:163 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:145;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:164 and a heavychain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:146; and those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:165 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:147.

In a third aspect, the ST2 antigen binding protein contains a lightchain variable domain comprising a) an LCDR1 having no more than threeamino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:106; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:117; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:128; b) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:107; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:118; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:129; c) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:108; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:119; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:130; d) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:109; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:120; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:131; e) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:110; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:121; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:132; f) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:111; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:122; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:133; g) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:112; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:123; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:134; h) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:113; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:124; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:135; i) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:114; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:125; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:136; j) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:115; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:126; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:137; k) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:116; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:127; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:138; l) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:166; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:169; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:172; m) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:167; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:170; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:173; or n) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:168; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:171; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:174; and a heavy chain variable domaincomprising o) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:40; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:51; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:62; p) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:41; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:52; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:63; q) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:42; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:53; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:64; r) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:43; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:54; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:65; s) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:44; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:55; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:66; t) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:45; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:56; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:67; u) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:46; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:57; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:68; v) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:47; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:58; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:69; w) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:48; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:59; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:70; x) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:49; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:60; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:71; y) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:50; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:61; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:72; z) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:148; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:151; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:154; aa) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:149; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:152; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:155; or bb) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:150; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:153; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:156.

Preferred ST2 antigen binding proteins of third aspect include thosecomprising the light chain variable domain of a) and the heavy chainvariable domain of o); those comprising the light chain variable domainof b) and the heavy chain variable domain of p); those comprising thelight chain variable domain of c) and the heavy chain variable domain ofq); those comprising the light chain variable domain of d) and the heavychain variable domain of r); those comprising the light chain variabledomain of e) and the heavy chain variable domain of s); those comprisingthe light chain variable domain of f) and the heavy chain variabledomain of t); those comprising the light chain variable domain of g) andthe heavy chain variable domain of u); those comprising the light chainvariable domain of h) and the heavy chain variable domain of v); thosecomprising the light chain variable domain of i) and the heavy chainvariable domain of w); those comprising the light chain variable domainof j) and the heavy chain variable domain of x); those comprising thelight chain variable domain of k) and the heavy chain variable domain ofy); those comprising the light chain variable domain of l) and the heavychain variable domain of z); those comprising the light chain variabledomain of m) and the heavy chain variable domain of aa); and thosecomprising the light chain variable domain of n) and the heavy chainvariable domain of bb).

In a fourth aspect of the invention, the ST2 antigen binding protein ofthe first, second, or third aspect binds to human ST2 with an affinityof less than or equal to 1×10⁻¹⁰ M.

In a fifth aspect of the invention, the ST2 antigen binding protein ofthe first, second, third, or fourth aspect inhibits binding of human ST2to human IL-33.

In a sixth aspect of the invention, the ST2 antigen binding protein ofthe first, second, third, fourth, or fifth aspect reduces humanIL-33-mediated ST2 signaling in human ST2-expressing cells.

In a seventh aspect of the invention, the ST2 antigen binding protein ofthe sixth aspect, reduces IL-33-mediated cynomolgus monkey ST2 signalingin cynomolgous monkey ST2-expressing cells.

In an eighth aspect of the invention, the ST2 antigen binding protein ofthe first, second, third, fourth, fifth, sixth or seventh aspect is anantibody, such as a human antibody. Preferred antibodies include thoseantibodies that comprise a light chain having the amino acid sequenceset forth in SEQ ID:84 and a heavy chain having the amino acid sequenceset forth in SEQ ID NO:18; those that comprise a light chain having theamino acid sequence set forth in SEQ ID:85 and a heavy chain having theamino acid sequence set forth in SEQ ID NO:19; those that comprise alight chain having the amino acid sequence set forth in SEQ ID:86 and aheavy chain having the amino acid sequence set forth in SEQ ID NO:20;those that comprise a light chain having the amino acid sequence setforth in SEQ ID:87 and a heavy chain having the amino acid sequence setforth in SEQ ID NO:21; those that comprise a light chain having theamino acid sequence set forth in SEQ ID:88 and a heavy chain having theamino acid sequence set forth in SEQ ID NO:22; those that comprise alight chain having the amino acid sequence set forth in SEQ ID:89 and aheavy chain having the amino acid sequence set forth in SEQ ID NO:23;those that comprise a light chain having the amino acid sequence setforth in SEQ ID:90 and a heavy chain having the amino acid sequence setforth in SEQ ID NO:24; those that comprise a light chain having theamino acid sequence set forth in SEQ ID:91 and a heavy chain having theamino acid sequence set forth in SEQ ID NO:25; those that comprise alight chain having the amino acid sequence set forth in SEQ ID:92 and aheavy chain having the amino acid sequence set forth in SEQ ID NO:26;those that comprise a light chain having the amino acid sequence setforth in SEQ ID:93 and a heavy chain having the amino acid sequence setforth in SEQ ID NO:27; those that comprise a light chain having theamino acid sequence set forth in SEQ ID:94 and a heavy chain having theamino acid sequence set forth in SEQ ID NO:28; those that comprise alight chain having the amino acid sequence set forth in SEQ ID: 160 anda heavy chain having the amino acid sequence set forth in SEQ ID NO:142;those that comprise a light chain having the amino acid sequence setforth in SEQ ID:161 and a heavy chain having the amino acid sequence setforth in SEQ ID NO:143; and those that comprise a light chain having theamino acid sequence set forth in SEQ ID: 162 and a heavy chain havingthe amino acid sequence set forth in SEQ ID NO:144.

In a ninth aspect, the invention provides isolated nucleic acidsencoding one or more polypeptide components of a ST2 antigen bindingprotein, e.g., an antibody light chain or antibody heavy chain. Inpreferred embodiments the nucleic acid encodes a polypeptide comprising:

a) a light chain variable domain having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ IDNO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:163, SEQID NO:164, or SEQ ID NO:165;

b) a heavy chain variable domain having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:145, SEQ IDNO:146, or SEQ ID NO:147;

c) a light chain variable domain having no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQID NO:104, SEQ ID NO:105, SEQ ID NO:163, SEQ ID NO:164, or SEQ IDNO:165;

d) a heavy chain variable domain having no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:145, SEQ ID NO:146, or SEQ ID NO:147;

e) a light chain variable domain comprising:

-   -   i) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:106; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:117; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:128;    -   ii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:107; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:118; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:129;    -   iii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:108; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:119; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:130;    -   iv) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:109; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:120; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:131;    -   v) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:110; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:121; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:132;    -   vi) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:111; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:122; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:133;    -   vii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:112; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:123; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:134;    -   viii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:113; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:124; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:135;    -   ix) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:114; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:125; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:136;    -   x) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:115; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:126; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:137;    -   xi) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:116; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:127; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:138;    -   xii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:166; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:169; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:172;    -   xiii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:167; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:170; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:173; or    -   xiv) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:168; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:171; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:174; or

f) a heavy chain variable domain comprising:

-   -   i) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:40; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:51; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:62;    -   ii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:41; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:52; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:63;    -   iii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:42; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:53; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:64;    -   iv) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:43; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:54; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:65;    -   v) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:44; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:55; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:66;    -   vi) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:45; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:56; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:67;    -   vii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:46; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:57; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:68;    -   viii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:47; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:58; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:69;    -   ix) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:48; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:59; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:70;    -   x) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:49; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:60; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:71;    -   xi) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:50; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:61; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:72;    -   xii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:148; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:151; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:154;    -   xiii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:149; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:152; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:155; or    -   xiv) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:150; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:153; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:156.

In certain embodiments of the ninth aspect, the polypeptide encodes anantibody light chain and is at least 80%, at least 90%, at least 95%, oris 100% identical to the nucleotide sequence set forth in SEQ ID NO:73,SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83,SEQ ID NO:157, SEQ ID NO:158, or SEQ ID NO:159. In other embodiments ofthe ninth aspect, the polypeptide encodes an antibody heavy chain and isat least 80%, at least 90%, at least 95%, or is 100% identical to thenucleotide sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:139, SEQ ID NO:140,or SEQ ID NO:141.

In a tenth aspect, the invention provides an expression vectorcomprising one or more isolated nucleic acids of the eighth aspect. Incertain embodiments, the expression vector encodes an antibody lightchain, an antibody heavy chain, or both an antibody light chain and aheavy chain.

In an eleventh aspect, the invention provides a recombinant host cellcomprising one or more isolated nucleic acids of the ninth aspectoperably linked to a promoter, including recombinant host cellscomprising one or more expression vectors of the tenth aspect of theinvention. In preferred embodiments, the recombinant host cell secretesan antibody that binds ST2. Preferred host cells are mammalian hostcells, including CHO cell lines.

In a twelfth aspect, the invention provides methods of treating anautoimmune or inflammatory disorder said method comprising administeringa therapeutically effective amount of a ST2 antigen binding protein ofany one of the first, second, third, fourth, fifth, sixth, sixth,seventh, or eighth aspects to a patient in need thereof. In preferredembodiments, the ST2 antigen binding protein is an antibody comprising alight chain variable domain amino acid sequence as set forth in SEQ IDNO:95 and a heavy chain variable domain amino acid sequence as set forthin SEQ ID NO:29 (e.g., Ab1), an antibody comprising a light chainvariable domain amino acid sequence as set forth in SEQ ID NO:96 and aheavy chain variable domain amino acid sequence as set forth in SEQ IDNO:30 (e.g., Ab2), an antibody comprising a light chain variable domainamino acid sequence as set forth in SEQ ID NO:97 and a heavy chainvariable domain amino acid sequence as set forth in SEQ ID NO:31 (e.g.,Ab3), an antibody comprising a light chain variable domain amino acidsequence as set forth in SEQ ID NO:98 and a heavy chain variable domainamino acid sequence as set forth in SEQ ID NO:32 (e.g., Ab4), anantibody comprising a light chain variable domain amino acid sequence asset forth in SEQ ID NO:99 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:33 (e.g., Ab5), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:100 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:34 (e.g., Ab6), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:101 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:35 (e.g., Ab7), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:102 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:36 (e.g., Ab8), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:103 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:37 (e.g., Ab9), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:104 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:38 (e.g., Ab10), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:105 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:39 (e.g., Ab11); an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:163 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:145 (e.g., Ab30); an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:164 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:146 (e.g., Ab32); or an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:165 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:147 (e.g., Ab33). In preferredembodiments, the ST2 antigen binding protein inhibits binding of IL-33to ST2. In particularly preferred embodiments, the autoimmune orinflammatory disorder is asthma, inflammatory bowel disease, rheumatoidarthritis, psoriasis, atopic dermatitis, fibrosis, chronic obstructivepulmonary disease, systemic lupus erythematosus, sclerosis, Wegener'sgranulomatosis, Behchet disease, rhinosinusitis, nasal polyposis,eosinophilic bronchitis, and cardiovascular disease.

In a thirteenth aspect, the invention provides a method of making an ST2antigen binding protein of any one of the first, second, third, fourth,fifth, sixth, sixth, seventh, or eighth aspects by culturing arecombinant host cell of the eleventh aspect and isolating the ST2antigen binding protein from said culture.

In a fourteenth aspect, the invention provides ST2 antigen bindingproteins of any one of the first, second, third, fourth, fifth, sixth,sixth, seventh, or eighth aspects that cross-compete with an antibodyselected from the group consisting of:

a) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:84 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:18;

b) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:85 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:19;

c) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:86 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:20;

d) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:87 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:21;

e) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:88 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:22;

f) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:89 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:23;

g) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:90 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:24;

h) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:91 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:25;

i) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:92 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:26;

j) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:93 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:27;

k) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:94 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:28;

l) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID: 160 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:142;

m) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID: 161 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:143; and

n) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID: 162 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:144.

In a fifteenth aspect, the invention provides an isolated ST2 antigenbinding protein, preferably an antibody or antigen binding fragmentthereof, that binds a polypeptide comprising human ST2 domain 1 anddomain 2 (SEQ ID NO:175), wherein binding is significantly inhibitedwhen a single mutation is introduced into human ST2 domain 1 or domain 2of the polypeptide, wherein the single mutation is selected from thegroup consisting of L14R, I15R, S33R, E43R, V47R, A62R, G65R, T79R,D92R, D97R, V104R, G138R, N152R, and V176R. By “significantly inhibited”it is meant that the measured difference in binding is statisticallysignificant. In preferred embodiments, binding is significantlyinhibited for two or more members of the group including all members ofthe group. In certain embodiments of the fifteenth aspect, binding alsois significantly activated when a single mutation is introduced intohuman ST2 domain 1 or domain 2 of the polypeptide, wherein the singlemutation is selected from the group consisting of L53R, R72A, and S73R.By “significantly activated” it is meant that the measured difference inbinding is statistically significant. In preferred embodiments, bindingis significantly activated for all members of the group. In certainembodiments of the fifteenth aspect, the ST2 binding proteincross-competes for binding to human ST2 with an antibody comprising alight chain comprising the amino acid sequence set forth in SEQ ID:85and a heavy chain comprising the amino acid sequence set forth in SEQ IDNO:19. In particularly preferred embodiments, the antigen bindingprotein is an antigen binding protein of the first, second, third,fourth, fifth, sixth, seventh, or eighth aspect.

In a sixteenth aspect, the invention provides an antigen binding proteinof the first, second, third, fourth, fifth, sixth, seventh, eighth,fourteenth, or fifteenth aspect wherein said ST2 binding proteinpreferably an antibody or antigen binding fragment thereof, binds aportion of ST2 comprising amino acids 33-44 and/or 88-94 of SEQ ID NO:1as determined by hydrogen/deuterium exchange analysis.

In a seventeenth aspect, the invention provides an antigen bindingprotein preferably an antibody or antigen binding fragment thereof, ofthe first, second, third, fourth, fifth, sixth, seventh, eighth,fourteenth, fifteenth, or sixteenth aspect which binds to ST2 creatingan interface, wherein the interface created by said binding comprises anST2 residue selected from the group consisting of K1, F2, P19, R20, Q21,G22, K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79, andY81. In preferred embodiments of the seventeenth aspect, the interfacecreated by said binding comprises ST2 residue P19, R20, Q21, G22, K23,and/or Y26, ST2 residue I70, V71, R72, S73, P74, T75, F76, N77, R78,T79, and/or Y81, or ST2 residues P19, R20, Q21, G22, K23, Y26, I70, V71,R72, S73, P74, T75, F76, N77, R78, T79, and Y81. The interface may bedetermined by solvent exposure difference between bound and unbound ST2and interface residues are defined as those amino acids having adifference greater than 10% and those that form water-mediated hydrogenbonds with said antibody or determined by as those amino acids having atleast one atom within 5 Å of the antibody.

In an eighteenth aspect, the invention provides an isolated ST2 antigenbinding protein, preferably an antibody or antigen binding fragmentthereof, comprising a) a light chain variable domain having at least 90%or at least 95% identity to the amino acid sequence set forth in SEQ IDNO:96, b) a heavy chain variable domain having at least 90% or at least95% identity to the amino acid sequence set forth in SEQ ID NO:30; c) alight chain variable domain of a) and a heavy chain variable domain ofb), d) a light chain variable domain having no more than ten or no morethan five amino acid additions, deletions or substitutions from theamino acid sequence set forth SEQ ID NO:96; e) a heavy chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:30; f) a light chain variable domain of d) and theheavy chain variable domain of e), g) a light chain variable domaincomprising an LCDR1 having no more than three amino acid additions,deletions, or substitutions from the LCDR1 sequence set forth in SEQ IDNO:107; an LCDR2 having no more than three amino acid additions,deletions, or substitutions from the LCDR2 sequence set forth in SEQ IDNO:118; and an LCDR3 having no more than three amino acid additions,deletions, or substitutions from the LCDR3 sequence set forth in SEQ IDNO:129, h) a heavy chain variable domain comprising an HCDR1 having nomore than three amino acid additions, deletions, or substitutions fromthe HCDR1 sequence set forth in SEQ ID NO:41; an HCDR2 having no morethan three amino acid additions, deletions, or substitutions from theHCDR2 sequence set forth in SEQ ID NO:52; and an HCDR3 having no morethan three amino acid additions, deletions, or substitutions from theHCDR3 sequence set forth in SEQ ID NO:63, or i) a light chain variabledomain of g) and the heavy chain variable domain of h).

In preferred embodiments of the eighteenth aspect, the light chainvariable region comprises D28 or a conservative substitution thereof,I29 or a conservative substitution thereof, S30 or a conservativesubstitution thereof, N31 or a conservative substitution thereof, Y32 ora conservative substitution thereof, Y49 or a conservative substitutionthereof, D50 or a conservative substitution thereof, N53 or aconservative substitution thereof, E55 or a conservative substitutionthereof, T56 or a conservative substitution thereof, D91 or aconservative substitution thereof, D92 or a conservative substitutionthereof, N93 or a conservative substitution thereof, F94 or aconservative substitution thereof, or L96 or a conservative substitutionthereof. In other preferred embodiments, the light chain variable regioncomprises D28 or a conservative substitution thereof, N31 or aconservative substitution thereof, D50 or a conservative substitutionthereof, N53 or a conservative substitution thereof, E55 or aconservative substitution thereof, D91 or a conservative substitutionthereof, and D92 or a conservative substitution thereof. In still otherpreferred embodiments, the light chain variable region comprises D28,N31, D50, N53, E55, D91, and D92.

The eighteenth aspect also includes ST2 binding proteins, preferablyantibodies or antigen binding fragment thereof, wherein the heavy chainvariable region comprises W33 or a conservative substitution thereof,I50 or a conservative substitution thereof, D57 or a conservativesubstitution thereof, R59 or a conservative substitution thereof, H99 ora conservative substitution thereof, G100 or a conservative substitutionthereof, T101 or a conservative substitution thereof, S102 or aconservative substitution thereof, S103 or a conservative substitutionthereof, D104 or a conservative substitution thereof, Y105 or aconservative substitution thereof, or Y106 or a conservativesubstitution thereof; wherein the heavy chain variable region comprisesS102 or a conservative substitution thereof, S103 or a conservativesubstitution thereof, D104 or a conservative substitution thereof, andY105 or a conservative substitution thereof; and wherein the heavy chainvariable region comprises S102, S103, D104, and Y105.

In certain embodiments of the eighteenth aspect, the ST2 antigen bindingprotein specifically binds human ST2 with an affinity of less than orequal to 1×10⁻¹⁰ M, inhibits binding of human ST2 to human IL-33,reduces human IL-33-mediated ST2 signaling in human ST2-expressingcells, inhibits binding of cynomolgus monkey ST2 to cynomolgus monkeyIL-33, reduces IL-33-mediated cynomolgus monkey ST2 signaling incynomolgus monkey ST2-expressing cells, and/or is an antibody such as ahuman antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 ST2 mAb treatment significantly inhibited IL-33-induced IL-5 inthe bronchoalveolar lavage fluid (BALF) Balb/c and C57B1/6 mice.

FIG. 2 ST2 mAb treatment is efficacious in a cockroach allergen(CRA)-induced model of asthma. ST2 antibody-treated mice hadsignificantly fewer BALF eosinophils than isotype control Ig-treatedmice.

FIG. 3 ST2 mAbs in the inhibition of human IL-33-induced IL-5 productionfrom CD4+ T cells from various donors. The (-) line depicts the positivecontrol value of human IL-33 in combination with human IL-2 withoutinhibition. The ( . . . . ) depicts the positive control value of humanIL-2. The (--) line depicts the media control value.

FIG. 4 Dose response of human IL-33 in human NK cell assay.

FIG. 5 Reduction of IL-33 activity in a human NK cell assay caused byAb2 versus commercially-available ST2 antibodies. Clones HB12, FB9 and2A5 were obtained from MBL International Corporation. Clone B4E6 wasobtained from MD Biosciences. Clone 97203 was obtained from R&D Systems.

FIG. 6 The location of the regions of ST2 bound by Ab2 as determined byHDX (see Example 12). The region corresponding to amino acids 15-26 ofthe extracellular domain of ST2 is highlighted in red and regioncorresponding to amino acids 70-76 of the extracellular domain of ST2 ishighlighted in magenta.

FIG. 7 Overall structure of ST2/Ab2 sc-dsFv complex. Two Ab2 sc-dsFvmolecules are shown in cartoon representation and colored in cyan/blueor light yellow/gold for light chain (LC)/heavy chain (HC) pairrespectively. Two ST2 molecules are shown in magenta and green cartoon.

FIG. 8 Binding interface. ST2 is shown in yellow cartoon. The heavychain and light chain of Ab2 are shown in grey and wheat cartoon. TheCDR loops for heavy chain and light chain are colored in the followingorder: CDR1: red(HC) or light red(LC); CDR2: green(HC) or light green(LC); and CDR3: blue(HC) or light blue (LC).

FIGS. 9A and 9B Electrostatic surface potential map of ST2 and Ab2sc-dsFv. FIG. 9A) Charge and surface complementarity of ST2 and Ab2sc-dsFv. The binding interface is highlighted in circle. FIG. 9B) Left:Ab2 (grey/wheat cartoon) binds to the positive-charged patch on ST2(surface); Right: ST2 (yellow cartoon) binds to the acidic patch of Ab2sc-dsFv (surface). For the electrostatic potential map, red surfacerepresents negative charge and blue surface represents positive charge.

FIG. 10 Residues within the Ab2 variable domains that form an interfacewith ST2 when bound to the antigen. The CDR regions are boxed. Residueswithin the interface are shown in bold. Residues that form hydrogenbonds or salt bridges with amino acids within ST2 are italicized.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited within the body of this specification are expresslyincorporated by reference in their entirety.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification, etc.Enzymatic reactions and purification techniques may be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manuel, 3^(rd) ed., Cold Spring Harbor Laboratory Press, coldSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,analytic chemistry, organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, chemicalanalyses, pharmaceutical preparation, formulation, and delivery andtreatment of patients.

ST2

The antigen binding proteins described herein bind to ST2. ST2 isexpressed as both a soluble non-signaling variant (soluble ST2 or sST2)and a full-length membrane-spanning form (FL ST2, ST2 or ST2L). Anexemplary human ST2L amino acid sequence is provided herein in Table 1.The protein is made up of several domains: Amino acids 1-18 ofcorrespond to the leader sequence which may be cleaved during processingof the protein in mammalian cells; amino acids 19-331 correspond to theextracellular domain; amino acids 332-350 correspond to thetransmembrane domain; and amino acids 351-556 correspond to theintracellular domain. In preferred embodiments, the antigen bindingprotein binds to the extracellular domain of ST2L and prevents theinteraction of ST2 with IL-33. An exemplary human IL-33 amino acidsequence is provided in Table 1.

IL-33 signals through a heterodimeric receptor comprising ST2L and AcP.An exemplary human AcP amino acid sequence is provided in Table 1. Thisprotein also is made up of several domains: Amino acids 1-20 correspondto the leader sequence which may be cleaved during processing of theprotein in mammalian cells; amino acids 21-367 correspond to theextracellular domain; amino acids 368-388 correspond to thetransmembrane domain; and amino acids 389-570 correspond to theintracellular domain. In exemplary embodiments, an ST2 antigen bindingprotein binds ST2L and prevents IL-33-mediated signaling in cellsexpressing ST2L and AcP.

TABLE 1 Human ST2 amino acid sequence (SEQ ID NO: 1)MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAXVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHSIYCIIAVCSVFLMLINVLVIILKMFWIEATLLWRDIAKPYKTRNDGKLYDAYVVYPRNYKSSTDGASRVEHFVHQILPDVLENKCGYTLCIYGRDMLPGEDVVTAVETNIRKSRRHIFILTPQITHNKEFAYEQEVALHCALIQNDAKVILIEMEALSELDMLQAEALQDSLQHLMKVQGTIKWREDHIANKRSLNSKFWKHVRYQMPVPSKIPRKASSLTPLAAQ KQHuman AcP amino acid sequence (SEQ ID NO: 2)MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVELACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETILDGKEYDIYVSYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKETKVKELKRAKTVLTVIKWKGEKSKYPQGRFWKQLQVAMPVKKSPRRSSSDEQGLSYSSLKNV Human IL-33 amino acid sequence (SEQ ID NO: 3)MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET X = E or A

Exemplary embodiments of the present invention bind both human andcynomolgus monkey ST2 with high affinity, including those that bind withhigh affinity and block interaction of cynomolgus monkey IL-33 tocynomolgus monkey ST2. These characteristics allow informativetoxicology studies in non-human primates.

An exemplary amino acid sequence of cynomolgus monkey ST2L is providedin Table 2. The protein is made up of several domains: amino acids 1-18of correspond to the leader sequence which may be cleaved duringprocessing of the protein in mammalian cells; amino acids 19-331correspond to the extracellular domain; amino acids 332-350 correspondto the transmembrane domain; and amino acids 351-556 correspond to theintracellular domain.

An exemplary amino acid sequences of cynomolgus monkey AcP is providedin Table 2. The protein is made up of several domains: amino acids 1-20of correspond to the leader sequence which may be cleaved duringprocessing of the protein in mammalian cells; amino acids 21-367correspond to the extracellular domain; amino acids 368-388 correspondto the transmembrane domain; and amino acids 389-570 correspond to theintracellular domain.

An exemplary amino acid sequence of cynomolgus monkey IL-33 is providedin Table 2.

TABLE 2 Cynomolgus monkey ST2 amino acid sequence (SEQ ID NO: 4)MGLWILAILTILVYSTAAKFSKQSWGLENEALIVRCPRQGKSSYIVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAEVADSGIYTCIVRSPTFNRTGYANVTIYKKQPDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYKAHKSFLVIDNVMTDDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSRFPVIRAPAHNETKEVEIGENTNLTCSACFGKGAQFLATVQWQLNGNKITDFSEPRIQQEEGQNQSFSNGLACVNTVLRIADVKEEDLLLRYDCLALNLHGLRRHTIRLSRKNPIDHQSTYCIIAVCSVLLMLINILVIILKTFWIEATLLWRDIAKPYKTRNDGKLYDAYVIYPRNYTSSADGASRVEYFVHQILPDVLENKCGYTLCIYGRDMLPGEDVVTAVETNIRKSRRHIFILTPQITHSEEFAYEQEVALHSALIQNDSKVILIEMEALSELDMLQAEALQDSLRHLMEVQGTIKWREDHVANKRSLNSKFWKHVRYQMPVPSKMPRKASSLTSLAAQKQCynomolgus monkey AcP amino acid sequence (SEQ ID NO: 5)MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIAFISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDIPIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAATVKQKVPAPRYTVELACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETILDGKEYDIYVSYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQGTQALLELKAGLENMASQGNINVILVQYKAVKETKVKELKRAKTVLTVIKWKGEKSKYPQGRFWKQLQVAMPVKKSPRRSSSDEQGLSYSSLKNVCynomolgus monkey IL-33 amino acid sequence (SEQ ID NO: 6)MKPKMKYSTNKISTAKRKNTASKALCFKLGKSQQKAKEVCHVYFMKLRSGLMIKKEACYFRRETTKRPSLKTGGKHKGHLVLAACQQQSTVECFAFGISGVPKYTRALHDSSITGISPITESLASLSTYNDQSITFALEDESYEIYVEDLKKDKKKDKVLLSYYESQHPSSESGDGVDGKMLMVTLSPTKDFWLQANNKEHSVELHKCEKPLPDQAFFVLHNRSFNCVSFECKTDPGVFIGVKDNHLALIKVDHSENLGSENILFKLSEI

ST2 Antigen Binding Proteins

The present invention provides antigen binding proteins thatspecifically bind ST2. Embodiments of antigen binding proteins comprisepeptides and/or polypeptides that specifically bind ST2. Such peptidesor polypeptides may optionally include one or more port-translationalmodifications. Embodiments of antigen binding proteins includeantibodies and fragments thereof, as variously defined herein, thatspecifically bind ST2. These include antibodies that specifically bindhuman ST2, including those that inhibit IL-33 from binding and/oractivating ST2.

The antigen binding proteins of the invention specifically bind to ST2.“Specifically binds” as used herein means that the antigen bindingprotein preferentially binds ST2 over other proteins. In someembodiments “specifically binds” means the ST2 antigen binding proteinhas a higher affinity for ST2 than for other proteins. ST2 antigenbinding proteins that specifically bind ST2 may have a binding affinityfor human ST2 of less than or equal to 1×10⁻⁷ M, less than or equal to2×10⁻⁷ M, less than or equal to 3×10⁻⁷ M, less than or equal to 4×10⁻⁷M, less than or equal to 5×10⁻⁷ M, less than or equal to 6×10⁻⁷ M, lessthan or equal to 7×10⁻⁷ M, less than or equal to 8×10⁻⁷ M, less than orequal to 9×10⁻⁷ M, less than or equal to 1×10⁻⁸ M, less than or equal to2×10⁻⁸ M, less than or equal to 3×10⁻⁸ M, less than or equal to 4×10⁻⁸M, less than or equal to 5×10⁻⁸ M, less than or equal to 6×10⁻⁸ M, lessthan or equal to 7×10⁻⁸ M, less than or equal to 8×10⁻⁸ M, less than orequal to 9×10⁻⁸ M, less than or equal to 1×10⁻⁹ M, less than or equal to2×10⁻⁹ M, less than or equal to 3×10⁻⁹ M, less than or equal to 4×10⁻⁹M, less than or equal to 5×10⁻⁹ M, less than or equal to 6×10⁻⁹ M, lessthan or equal to 7×10⁻⁹ M, less than or equal to 8×10⁻⁹ M, less than orequal to 9×10⁻⁹ M, less than or equal to 1×10⁻¹⁰ M, less than or equalto 2×10⁻¹⁰ M, less than or equal to 3×10⁻¹⁰ M, less than or equal to4×10⁻¹⁰ M, less than or equal to 5×10⁻¹⁰ M, less than or equal to6×10⁻¹⁰ M, less than or equal to 7×10⁻¹⁰ M, less than or equal to8×10⁻¹⁰ M, less than or equal to 9×10⁻¹⁰ M, less than or equal to1×10⁻¹¹ M, less than or equal to 2×10⁻¹¹ M, less than or equal to3×10⁻¹¹ M, less than or equal to 4×10⁻¹¹ M, less than or equal to5×10⁻¹¹ M, less than or equal to 6×10⁻¹¹ M, less than or equal to7×10⁻¹¹ M, less than or equal to 8×10⁻¹¹ M, less than or equal to9×10⁻¹¹ M, less than or equal to 1×10⁻¹² M, less than or equal to2×10⁻¹² M, less than or equal to 3×10⁻¹² M, less than or equal to4×10⁻¹² M, less than or equal to 5×10⁻¹² M, less than or equal to6×10⁻¹² M, less than or equal to 7×10⁻¹² M, less than or equal to8×10⁻¹² M, or less than or equal to 9×10⁻¹² M.

Methods of measuring the binding affinity of an antigen binding proteinare well known in the art. Methods in common use for affinitydetermination include Surface Plasmon Resonance (SPR) (Morton and Myszka“Kinetic analysis of macromolecular interactions using surface plasmonresonance biosensors” Methods in Enzymology (1998) 295, 268-294),Bio-Layer Interferometry, (Abdiche et al “Determining Kinetics andAffinities of Protein Interactions Using a Parallel Real-time Label-freeBiosensor, the Octet” Analytical Biochemistry (2008) 377, 209-217),Kinetic Exclusion Assay (KinExA) (Darling and Brault “Kinetic exclusionassay technology: characterization of molecular interactions” Assay andDrug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al“Isothermal Titration Calorimetry of Protein-Protein Interactions”Methods (1999) 19, 213-221) and analytical ultracentrifugation (Lebowitzet al “Modern analytical ultracentrifugation in protein science: Atutorial review” Protein Science (2002), 11:2067-2079). Example 3provides exemplary methods.

It is understood that when reference is made to the various embodimentsof the ST2-binding antibodies herein, that it also encompassesST2-binding fragments thereof. An ST2-binding fragment comprises any ofthe antibody fragments or domains described herein that retains theability to specifically bind to ST2. The ST2-binding fragment may be inany of the scaffolds described herein.

In certain therapeutic embodiments, an ST2 antigen binding proteininhibits binding of ST2 to IL-33 and/or inhibits one or more biologicalactivities associated with the binding of ST2 to IL-33, e.g.,IL-33-mediated signaling. Such antigen binding proteins are said to be“neutralizing.” In certain embodiments, the neutralizing ST2 antigenbinding protein specifically binds ST2 and inhibits binding of ST2 toIL-33 from anywhere between 10% to 100%, such as by at least about 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99% or more. For example, ST2 antigen bindingproteins may be tested for neutralizing ability by determining theability of the antigen binding protein to block binding of IL-33 to ST2or IL-33 to co-receptors ST2 and AcP, see, e.g., the IL-33 blockingassays of Example 6. Alternatively, ST2 antigen binding proteins may betested for neutralizing ability in an assay that measures the effect ofthe presence of the ST2 antigen binding protein in an assay measuring anIL-33 mediated biological function. For example, the ability of IL-33 toinduce a biological response, such as intracellular signaling orincreased mRNA expression of mediators or secretion of mediators such ascytokines and chemokines from cells such as eosinophils, basophils, Tcells, mast cells, NK cells, NKT cells, neutrophils, or innate helpercells. Alternatively, the ability of IL-33 to promote thedifferentiation, proliferation, survival, chemotaxis, shape change oradhesive properties of cells such as eosinophils, basophils, T cells,mast cells, NK cells, NKT cells, neutrophils, or innate helper cells.Alternatively, the ability of IL-33 to induce cell surface expression ofcertain markers of cell activation, such as CD11b, on cells such aseosinophils, basophils, T cells, mast cells, NK cells, NKT cells,neutrophils, or innate helper cells. Exemplary methods are provided inExamples 7-10.

Embodiments of antigen binding proteins comprise a scaffold structure,as variously defined herein, with one or more complementaritydetermining regions (CDRs). Embodiments further include antigen bindingproteins comprising a scaffold structure with one or more antibodyvariable domains, either heavy or light. Embodiments include antibodiesthat comprise a light chain variable domain selected from the groupconsisting of Ab1 Light Chain Variable Domain (LCv), Ab2 LCv, Ab3 LCv,Ab4 LCv, Ab5 LCv, Ab6 LCv, Ab7 LCv, Ab8 LCv, Ab9 LCv, Ab10LCv, Ab11 LCv,Ab30 LCv, Ab32 LCv, and Ab33 LCv (SEQ ID NO:95-105, 163-165,respectively) and/or a heavy chain variable domain selected from thegroup consisting of Ab1 Heavy Chain Variable Domain (HCv), Ab2 HCv, Ab3HCv, Ab4 HCv, Ab5 HCv, Ab6 HCv, Ab7 HCv, Ab8 HCv, Ab9 HCv, Ab10 HCv,Ab11HCv, Ab30HCv, Ab32HCv, and Ab33HCv (SEQ ID NO:29-39, 145-147,respectively), and fragments, derivatives, muteins, and variantsthereof.

An exemplary light chain comprising Ab1 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:84.

An exemplary light chain comprising Ab2 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:85.

An exemplary light chain comprising Ab3 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:86.

An exemplary light chain comprising Ab4 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:87.

An exemplary light chain comprising Ab5 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:88.

An exemplary light chain comprising Ab6 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:89.

An exemplary light chain comprising Ab7 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:90.

An exemplary light chain comprising Ab8 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:91.

An exemplary light chain comprising Ab9 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:92.

An exemplary light chain comprising Ab10 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:93.

An exemplary light chain comprising Ab11 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:94.

An exemplary light chain comprising Ab30 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:160.

An exemplary light chain comprising Ab32 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:161.

An exemplary light chain comprising Ab33 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:162.

An exemplary heavy chain comprising Ab1 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:18.

An exemplary heavy chain comprising Ab2 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:19.

An exemplary heavy chain comprising Ab3 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:20.

An exemplary heavy chain comprising Ab4 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:21.

An exemplary heavy chain comprising Ab5 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:22.

An exemplary heavy chain comprising Ab6 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:23.

An exemplary heavy chain comprising Ab7 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:24.

An exemplary heavy chain comprising Ab8 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:25.

An exemplary heavy chain comprising Ab9 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:26.

An exemplary heavy chain comprising Ab10 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:27.

An exemplary heavy chain comprising Ab11 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:28.

An exemplary heavy chain comprising Ab30 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:142.

An exemplary heavy chain comprising Ab32 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:143.

An exemplary heavy chain comprising Ab33 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:144.

Additional examples of scaffolds that are envisioned include:fibronectin, neocarzinostatin CBM4-2, lipocalins, T-cell receptor,protein-A domain (protein Z), Im9, TPR proteins, zinc finger domains,pVIII, avian pancreatic polypeptide, GCN4, WW domain Src homology domain3, PDZ domains, TEM-1 beta-lactamase, thioredoxin, staphylococcalnuclease, PHD-finger domains, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-D1,LDTI, MTI-II, scorpion toxins, insect defensin-A peptide, EETI-II,Min-23, CBD, PBP, cytochrome b-562, Ld1 receptor domains,gamma-crystallin, ubiquitin, transferrin, and or C-type lectin-likedomains. Non-antibody scaffolds and their use as therapeutics arereviewed in Gebauer and Skerra, Curr. Opin. Chem. Biol., 13:245-255(2009) and Binz et al., Nat. Biotech., 23(10): 1257-1268 (2005), whichare incorporated herein by reference in its entirety.

Aspects of the invention include antibodies comprising the followingvariable domains: Ab1 LCv/Ab1 HCv (SEQ ID NO:95/SEQ ID NO:29), Ab2LCv/Ab2 HCv (SEQ ID NO:96/SEQ ID NO:30), Ab3 LCv/Ab3 HCv (SEQ IDNO:97/SEQ ID NO:31), Ab4 LCv/Ab4 HCv (SEQ ID NO:98/SEQ ID NO:32), Ab5LCv/Ab5 HCv (SEQ ID NO:99/SEQ ID NO:33), Ab6 LCv/Ab6 HCv (SEQ IDNO:100/SEQ ID NO:34), Ab7 LCv/Ab7 HCv (SEQ ID NO:101/SEQ ID NO:35), Ab8LCv/Ab8 HCv (SEQ ID NO:102/SEQ ID NO:36), Ab9 LCv/Ab9 HCv (SEQ IDNO:103/SEQ ID NO:37), Ab10 LCv/Ab10 HCv (SEQ ID NO:104/SEQ ID NO:38),Ab11 LCv/Ab11 HCv (SEQ ID NO:105/SEQ ID NO:39), Ab30 LCv/Ab30 HCv (SEQID NO:163/SEQ ID NO:145), Ab32 LCv/Ab32 HCv (SEQ ID NO:164/SEQ IDNO:146), Ab33 LCv/Ab33 HCv (SEQ ID NO:165/SEQ ID NO:147), andcombinations thereof, as well as fragments, derivatives, muteins andvariants thereof.

Exemplary antibodies of the invention include Ab1 (SEQ ID NO:84/SEQ IDNO:18), Ab2 (SEQ ID NO:85/SEQ ID NO:19), Ab3 (SEQ ID NO:86/SEQ IDNO:20), Ab4 (SEQ ID NO:87/SEQ ID NO:21), Ab5 (SEQ ID NO:88/SEQ IDNO:22), Ab6 (SEQ ID NO:89/SEQ ID NO:23), Ab7 (SEQ ID NO:90/SEQ IDNO:24), Ab8 (SEQ ID NO:91/SEQ ID NO:25), Ab9 (SEQ ID NO:92/SEQ IDNO:26), Ab10 (SEQ ID NO:93/SEQ ID NO:27), Ab11 (SEQ ID NO:94/SEQ IDNO:28), Ab30 (SEQ ID NO:160/SEQ ID NO:142), Ab32 (SEQ ID NO:161/SEQ IDNO:143), and Ab33 (SEQ ID NO:162/SEQ ID NO:144).

Typically, each variable domain of an antibody light or heavy chaincomprises three CDRs. The heavy chain variable domain comprises a heavychain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3(HCDR3). The light chain variable domain comprises a light chain CDR1(LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). Incertain embodiments, an antigen binding protein comprises one or moreCDRs contained within the preferred variable domains described herein.

Examples of such CDRs include, but are not limited to:

the CDRs of Ab1 LCv: LCDR1 (SEQ ID NO:106), LCDR2 (SEQ ID NO:117), andLCDR3 (SEQ ID NO:128);

the CDRs of Ab2 LCv: LCDR1 (SEQ ID NO:107), LCDR2 (SEQ ID NO:118), andLCDR3 (SEQ ID NO:129);

the CDRs of Ab3 LCv: LCDR1 (SEQ ID NO:108), LCDR2 (SEQ ID NO:119), andLCDR3 (SEQ ID NO:130);

the CDRs of Ab4 LCv: LCDR1 (SEQ ID NO:109), LCDR2 (SEQ ID NO:120), andLCDR3 (SEQ ID NO:131);

the CDRs of Ab5 LCv: LCDR1 (SEQ ID NO:110), LCDR2 (SEQ ID NO:121), andLCDR3 (SEQ ID NO:132);

the CDRs of Ab6 LCv: LCDR1 (SEQ ID NO:111), LCDR2 (SEQ ID NO:122), andLCDR3 (SEQ ID NO:133);

the CDRs of Ab7 LCv: LCDR1 (SEQ ID NO:112), LCDR2 (SEQ ID NO:123), andLCDR3 (SEQ ID NO:134);

the CDRs of Ab8 LCv: LCDR1 (SEQ ID NO:113), LCDR2 (SEQ ID NO:124), andLCDR3 (SEQ ID NO:135);

the CDRs of Ab9 LCv: LCDR1 (SEQ ID NO:114), LCDR2 (SEQ ID NO:125), andLCDR3 (SEQ ID NO:136);

the CDRs of Ab10 LCv: LCDR1 (SEQ ID NO:115), LCDR2 (SEQ ID NO:126), andLCDR3 (SEQ ID NO:137);

the CDRs of Ab11 LCv: LCDR1 (SEQ ID NO:116), LCDR2 (SEQ ID NO:127), andLCDR3 (SEQ ID NO:138);

the CDRs of Ab30 LCv: LCDR1 (SEQ ID NO:166), LCDR2 (SEQ ID NO:169), andLCDR3 (SEQ ID NO:172);

the CDRs of Ab32 LCv: LCDR1 (SEQ ID NO:167), LCDR2 (SEQ ID NO:170), andLCDR3 (SEQ ID NO:173);

the CDRs of Ab33 LCv: LCDR1 (SEQ ID NO:168), LCDR2 (SEQ ID NO:171), andLCDR3 (SEQ ID NO:174);

the CDRs of Ab1 HCv: HCDR1 (SEQ ID NO:40), HCDR2 (SEQ ID NO:51), andHCDR3 (SEQ ID NO:62);

the CDRs of Ab2 HCv: HCDR1 (SEQ ID NO:41), HCDR2 (SEQ ID NO:52), andHCDR3 (SEQ ID NO:63);

the CDRs of Ab3 HCv: HCDR1 (SEQ ID NO:42), HCDR2 (SEQ ID NO:53), andHCDR3 (SEQ ID NO:64);

the CDRs of Ab4 HCv: HCDR1 (SEQ ID NO:43), HCDR2 (SEQ ID NO:54), andHCDR3 (SEQ ID NO:65);

the CDRs of Ab5 HCv: HCDR1 (SEQ ID NO:44), HCDR2 (SEQ ID NO:55), andHCDR3 (SEQ ID NO:66);

the CDRs of Ab6 HCv: HCDR1 (SEQ ID NO:45), HCDR2 (SEQ ID NO:56), andHCDR3 (SEQ ID NO:67);

the CDRs of Ab7 HCv: HCDR1 (SEQ ID NO:46), HCDR2 (SEQ ID NO:57), andHCDR3 (SEQ ID NO:68);

the CDRs of Ab8 HCv: HCDR1 (SEQ ID NO:47), HCDR2 (SEQ ID NO:58), andHCDR3 (SEQ ID NO:69);

the CDRs of Ab9 HCv: HCDR1 (SEQ ID NO:48), HCDR2 (SEQ ID NO:59), andHCDR3 (SEQ ID NO:70);

the CDRs of Ab10 HCv: HCDR1 (SEQ ID NO:49), HCDR2 (SEQ ID NO:60), andHCDR3 (SEQ ID NO:71);

the CDRs of Ab11 HCv: HCDR1 (SEQ ID NO:50), HCDR2 (SEQ ID NO:61), andHCDR3 (SEQ ID NO:72);

the CDRs of Ab30 HCv: HCDR1 (SEQ ID NO:148), HCDR2 (SEQ ID NO:151), andHCDR3 (SEQ ID NO:154);

the CDRs of Ab32 HCv: HCDR1 (SEQ ID NO:149), HCDR2 (SEQ ID NO:152), andHCDR3 (SEQ ID NO:155); and

the CDRs of Ab33 HCv: HCDR1 (SEQ ID NO:150), HCDR2 (SEQ ID NO:153), andHCDR3 (SEQ ID NO:156).

In some embodiments, the antigen binding protein comprises: A) apolypeptide, e.g., a light chain, that comprises an LCDR1 having anamino acid sequence selected from the group consisting of SEQ IDNOS:106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 166, 167, and168; an LCDR2 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 169, 170, and 171; and/or an LCDR3 having an amino acidsequence selected from the group consisting of SEQ ID NOS:128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 172, 173, and 174; and/or B) apolypeptide, e.g., a heavy chain, that comprises an HCDR1 having anamino acid sequence selected from the group consisting of SEQ ID NOS:40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 148, 149, and 150; an HCDR2having an amino acid sequence selected from the group consisting of SEQID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 151, 152, and 153;and/or an HCDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,154, 155, and 156.

In further embodiments, the antigen binding protein comprise A) a lightchain amino acid sequence that comprises a LCDR1, LCDR2, and LCDR3 ofany of Ab1 LCv, Ab2 LCv, Ab3 LCv, Ab4 LCv, Ab5 LCv, Ab6 LCv, Ab7 LCv,Ab8 LCv, Ab9 LCv, Ab10 LCv, Ab11 LCv, Ab30 LCv, Ab32 LCv, and Ab33 LCvand B) a heavy chain amino acid sequence that comprises a HCDR1, HCDR2,and HCDR3 of any of Ab1 HCv, Ab2 HCv, Ab3 HCv, Ab4 HCv, Ab5 HCv, Ab6HCv, Ab7 HCv, Ab8 HCv, Ab9 HCv, Ab10 HCv, Ab11 HCv, Ab30 HCv, Ab32 HCv,and Ab33 HCv.

In certain embodiments, the CDRs include no more than one, no more thantwo, no more than three, no more than four, no more than five, or nomore than six amino acid additions, deletions, or substitutions from anexemplary CDR set forth herein.

Aspects of the invention include antibodies comprising a light chainvariable domain selected from the group consisting of SEQ ID NOS:95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164, and 165. Aspects ofthe invention include antibodies comprising a heavy chain variabledomain selected from the group consisting of SEQ ID NOS:29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 145, 146, and 147. Further aspects of theinvention include antibodies comprising A) a light chain variable domainselected from the group consisting of SEQ ID NOS: 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 163, 164, and 165, and B) a heavy chainvariable domain selected from the group consisting of SEQ ID NOS: 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 145, 146, and 147.

Antibodies of the invention can comprise any constant region known inthe art. The light chain constant region can be, for example, a kappa-or lambda-type light chain constant region, e.g., a human kappa- orlambda-type light chain constant region. The heavy chain constant regioncan be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-typeheavy chain constant region, e.g., a human alpha-, delta-, epsilon-,gamma-, or mu-type heavy chain constant region. In one embodiment thelight or heavy chain constant region is a fragment, derivative, variant,or mutein of a naturally occurring constant region.

Aspects of the invention include antibodies comprising a light chainvariable region selected from the group consisting of SEQ ID NOS: 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164, and 165 havingno more than one, no more than two, no more than three, no more thanfour, no more than five, no more than six, no more than seven, no morethan eight, no more than nine, or no more than ten amino acid additions,deletions, or substitutions. Aspects of the invention include antibodiescomprising a heavy chain variable region selected from the groupconsisting of SEQ ID NOS: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,145, 146, and 147 having no more than one, no more than two, no morethan three, no more than four, no more than five, no more than six, nomore than seven, no more than eight, no more than nine, or no more thanten amino acid additions, deletions, or substitutions. Further aspectsof the invention include antibodies comprising A) comprising a lightchain variable region selected from the group consisting of SEQ ID NOS:95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 163, 164, and 165having no more than one, no more than two, no more than three, no morethan four, no more than five, no more than six, no more than seven, nomore than eight, no more than nine, or no more than ten amino acidadditions, deletions, or substitutions, and B) a heavy chain variableregion selected from the group consisting of SEQ ID NOS: 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 145, 146, and 147 having no more than one,no more than two, no more than three, no more than four, no more thanfive, no more than six, no more than seven, no more than eight, no morethan nine, or no more than ten amino acid additions, deletions, orsubstitutions.

In one variation, the antigen binding protein comprises an amino acidsequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a light chain variable region amino acid sequenceselected from the group consisting of SEQ ID NOS: 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 163, 164, and 165. In another variation,the antigen binding protein comprises an amino acid sequence that is atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a heavy chain variable region amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 145, 146, and 147. In yet a further embodiment, the antigenbinding protein comprises A) an amino acid sequence that is at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto a light chain variable region amino acid sequence selected from thegroup consisting of SEQ ID NOS: 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 163, 164, and 165, and B) an amino acid sequence that is atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a heavy chain variable region amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 145, 146, and 147.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR3. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 172, 173, 174, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 154, 155, and 156. In certain embodiments, the amino acidsequence includes no more than one, no more than two, no more thanthree, no more than four, no more than five, or no more than six aminoacid additions, deletions, or substitutions from the exemplary sequenceset forth in SEQ ID NOS:128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 172, 173, 174, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,154, 155, and 156. Thus, embodiments of the invention include antigenbinding protein comprising an amino acid sequence that is at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence selected from the group of sequences set forth inSEQ ID NOS:128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 172,173, 174, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 154, 155, and 156.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR2. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 169, 170, 171, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 151, 152, and 153. In certain embodiments, the amino acidsequence includes no more than one, no more than two, no more thanthree, no more than four, no more than five, or no more than six aminoacid additions, deletions, or substitutions from the exemplary sequenceset forth in SEQ ID NOS:117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 169, 170, 171, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,151, 152, and 153. Thus, embodiments of the invention include antigenbinding protein comprising an amino acid sequence that is at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence selected from the group of sequences set forth inSEQ ID NOS:117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 169,170, 171, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 151, 152, and 153.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR1. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 166, 167, 168, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 148, 149, and 150. In certain embodiments, the amino acidsequence includes no more than one, no more than two, no more thanthree, no more than four, no more than five, or no more than six aminoacid additions, deletions, or substitutions from the exemplary sequenceset forth in SEQ ID NOS:106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 166, 167, 168, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,148, 149, and 150. Thus, embodiments of the invention include antigenbinding protein comprising an amino acid sequence that is at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence selected from the group of sequences set forth inSEQ ID NOS:106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 166,167, 168, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 148, 149, and 150.

The antigen binding proteins of the invention comprise the scaffolds oftraditional antibodies, including human and monoclonal antibodies,bispecific antibodies, diabodies, minibodies, domain antibodies,synthetic antibodies (sometimes referred to herein as “antibodymimetics”), chimeric antibodies, antibody fusions (sometimes referred toas “antibody conjugates”), and fragments of each, respectively. Theabove described CDRs, including various combinations of the CDRs, may begrafted into any of the following scaffolds.

As used herein, the term “antibody” refers to the various forms ofmonomeric or multimeric proteins comprising one or more polypeptidechains that specifically binds to an antigen, as variously describedherein. In certain embodiments, antibodies are produced by recombinantDNA techniques. In additional embodiments, antibodies are produced byenzymatic or chemical cleavage of naturally occurring antibodies. Inanother aspect, the antibody is selected from the group consisting of:a) a human antibody; b) a humanized antibody; c) a chimeric antibody; d)a monoclonal antibody; e) a polyclonal antibody; f) a recombinantantibody; g) an antigen-binding fragment; h) a single chain antibody; i)a diabody; j) a triabody, k) a tetrabody, l) a Fab fragment; m) aF(ab′)₂ fragment, n) an IgA antibody, o) an IgD antibody, p) an IgEantibody, q) an IgG1 antibody, r) an IgG2 antibody, s) an IgG3 antibody,t) an IgG4 antibody, and u) an IgM antibody.

A variable region or domain comprises at least three heavy or lightchain CDRs embedded within a framework region (designated frameworkregions FR1, FR2, FR3, and FR4). Kabat et al., 1991, Sequences ofProteins of Immunological Interest, Public Health Service N.I.H.,Bethesda, Md. Traditional antibody structural units typically comprise atetramer. Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” and one “heavy” chain.The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subclasses, including, but not limited to IgG1, IgG2, IgG3, andIgG4. IgM has subclasses, including, but not limited to IgM1 and IgM2.Embodiments of the invention include all such classes and subclasses ofantibodies that incorporate a variable domain or CDR of the antigenbinding proteins, as described herein.

Some naturally occurring antibodies, such as those found in camels andllamas, are dimers consisting of two heavy chains and include no lightchains. The invention encompasses dimeric antibodies of two heavychains, or fragments thereof, that can bind to ST2.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three hypervariable regions, i.e., the complementaritydetermining regions or CDRs. The CDRs are primarily responsible forantigen recognition and binding. The CDRs from the two chains of eachpair are aligned by the framework regions, enabling binding to aspecific epitope. From N-terminal to C-terminal, both light and heavychains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.The assignment of amino acids to each domain is in accordance with thedefinitions of Kabat.

CDRs constitute the major surface contact points for antigen binding.The CDR3 or the light chain and, particularly, CDR3 of the heavy chainmay constitute the most important determinants in antigen binding withinthe light and heavy chain variable regions. In some antibodies, theheavy chain CDR3 appears to constitute the major area of contact betweenthe antigen and the antibody. In vitro selection schemes in which CDR3alone is varied can be used to vary the binding properties of anantibody or determine which residues contribute to the binding of anantigen.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is typically not present in the mature antibody. Apolynucleotide encoding an antibody of the invention may encode anaturally occurring a signal sequence or a heterologous signal sequenceas described below.

In one embodiment, the antigen binding protein is an antibody comprisingfrom one to six of the exemplary CDRs described herein. The antibodiesof the invention may be of any type including IgM, IgG (including IgG1,IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. In a specific embodimentthe antigen binding protein is an IgG type antibody, e.g., a IgG1antibody.

In some embodiments, for example when the antigen binding protein is anantibody with complete heavy and light chains, the CDRs are all from thesame species, e.g., human. Alternatively, for example in embodimentswherein the antigen binding protein contains less than six CDRs from thesequences outlined above, additional CDRs may be either from otherspecies or may be different human CDRs than those depicted in theexemplary sequences. For example, HCDR3 and LCDR3 regions from theappropriate sequences identified herein may be used with HCDR1, HCDR2,LCDR1, and LCDR2 being optionally selected from alternate species ordifferent human antibody sequences, or combinations thereof. Forexample, the CDRs of the invention can replace the CDR regions ofcommercially relevant chimeric or humanized antibodies.

Specific embodiments utilize scaffold components of the antigen bindingproteins that are human components. In some embodiments, however, thescaffold components can be a mixture from different species. As such, ifthe antigen binding protein is an antibody, such antibody may be achimeric antibody and/or humanized antibody. In general, both “chimericantibodies” and humanized antibodies” refer to antibodies that combineregions from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse (or rat, in somecases) and the constant region(s) from a human.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except one or more CDRs, is encoded by a polynucleotide ofhuman origin or is identical to such an antibody except within one ormore CDRs. The CDRs, some or all of which are encoded by nucleic acidsoriginating in a non-human organism, are grafted into the beta-sheetframework of a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones 1986,Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. In the exemplary embodiments described herein, theidentified CDRs are human, and thus both humanized and chimericantibodies in this context include some non-human CDRs; for example,humanized antibodies may be generated that comprise the HCDR3 and LCDR3regions, with one or more of the other CDR regions being of a differentspecies origin.

In one embodiment, the ST2 antigen binding protein is a multispecificantibody, and notably a bispecific antibody, also sometimes referred toas “diabodies.” These are antibodies that bind to two or more differentantigens or different epitopes on a single antigen. In certainembodiments, a bispecific antibody binds ST2 and an antigen on a humaneffector cell (e.g., T cell). Such antibodies are useful in targeting aneffector cell response against a ST2 expressing cells, such as anST2-expressing tumor cell. In preferred embodiments, the human effectorcell antigen is CD3. U.S. Pat. No. 7,235,641. Methods of makingbispecific antibodies are known in the art. One such method involvesengineering the Fc portion of the heavy chains such as to create “knobs”and “holes” which facilitate heterodimer formation of the heavy chainswhen co-expressed in a cell. U.S. Pat. No. 7,695,963. Another methodalso involves engineering the Fc portion of the heavy chain but useselectrostatic steering to encourage heterodimer formation whilediscouraging homodimer formation of the heavy chains when co-expressedin a cell. WO 09/089,004, which is incorporated herein by reference inits entirety.

In one embodiment, the ST2 antigen binding protein is a minibody.Minibodies are minimized antibody-like proteins comprising a scFv joinedto a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061.

In one embodiment, the ST2 antigen binding protein is a domain antibody;see, for example U.S. Pat. No. 6,248,516. Domain antibodies (dAbs) arefunctional binding domains of antibodies, corresponding to the variableregions of either the heavy (VH) or light (VL) chains of humanantibodies. dABs have a molecular weight of approximately 13 kDa, orless than one-tenth the size of a full antibody. dABs are well expressedin a variety of hosts including bacterial, yeast, and mammalian cellsystems. In addition, dAbs are highly stable and retain activity evenafter being subjected to harsh conditions, such as freeze-drying or heatdenaturation. See, for example, U.S. Pat. Nos. 6,291,158; 6,582,915;6,593,081; 6,172,197; US Serial No. 2004/0110941; European Patent0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 andWO03/002609.

In one embodiment, the ST2 antigen binding protein is an antibodyfragment, that is a fragment of any of the antibodies outlined hereinthat retain binding specificity to ST2. In various embodiments, theantibody binding proteins comprise, but are not limited to, a F(ab),F(ab′), F(ab′)2, Fv, or a single chain Fv fragments. At a minimum, anantibody, as meant herein, comprises a polypeptide that can bindspecifically to ST2 comprising all or part of a light or heavy chainvariable region, such as one or more CDRs.

Further examples of ST2-binding antibody fragments include, but are notlimited to, (i) the Fab fragment consisting of VL, VH, CL and CH1domains, (ii) the Fd fragment consisting of the VH and CH1 domains,(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546)which consists of a single variable, (v) isolated CDR regions, (vi)F(ab)₂ fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988,Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:5879-5883), (viii) bispecific single chain Fv dimers (PCT/US92/09965)and (ix) “diabodies” or “triabodies”, multivalent or multispecificfragments constructed by gene fusion (Tomlinson et. al., 2000, MethodsEnzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl.Acad. Sci. U.S.A. 90:6444-6448). The antibody fragments may be modified.For example, the molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al., 1996,Nature Biotech. 14:1239-1245). Aspects of the invention includeembodiments wherein the non-CDR components of these fragments are humansequences.

In one embodiment, the ST2 antigen binding protein is a fully humanantibody. In this embodiment, as outlined above, specific structurescomprise complete heavy and light chains depicted comprising the CDRregions. Additional embodiments utilize one or more of the CDRs of theinvention, with the other CDRs, framework regions, J and D regions,constant regions, etc., coming from other human antibodies. For example,the CDRs of the invention can replace the CDRs of any number of humanantibodies, particularly commercially relevant antibodies

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87. Single chain antibodiesderived from antibodies provided herein (including but not limited toscFvs comprising the variable domain combinations of Ab1 LCv/Ab1 HCv(SEQ ID NO:95/SEQ ID NO:29), Ab2 LCv/Ab2 HCv (SEQ ID NO:96/SEQ IDNO:30), Ab3 LCv/Ab3 HCv (SEQ ID NO:97/SEQ ID NO:31), Ab4 LCv/Ab4 HCv(SEQ ID NO:98/SEQ ID NO:32), Ab5 LCv/Ab5 HCv (SEQ ID NO:99/SEQ IDNO:33), Ab6 LCv/Ab6 HCv (SEQ ID NO:100/SEQ ID NO:34), Ab7 LCv/Ab7 HCv(SEQ ID NO:101/SEQ ID NO:35), Ab8 LCv/Ab8 HCv (SEQ ID NO:102/SEQ IDNO:36), Ab9 LCv/Ab9 HCv (SEQ ID NO:103/SEQ ID NO:37), Ab10 LCv/Ab10 HCv(SEQ ID NO:104/SEQ ID NO:38), and Ab11 LCv/Ab11 HCv (SEQ ID NO:105/SEQID NO:39), Ab30 LCv/Ab30 HCv (SEQ ID NO:163/SEQ ID NO:145), Ab32LCv/Ab32 HCv (SEQ ID NO:164/SEQ ID NO:146), Ab33 LCv/Ab33 HCv (SEQ IDNO:165/SEQ ID NO:147), and combinations thereof are encompassed by thepresent invention.

In one embodiment, the ST2 antigen binding protein is an antibody fusionprotein (sometimes referred to herein as an “antibody conjugate”). Theconjugate partner can be proteinaceous or non-proteinaceous; the lattergenerally being generated using functional groups on the antigen bindingprotein and on the conjugate partner. In certain embodiments, theantibody is conjugated to a non-proteinaceous chemical (drug) to form anantibody drug conjugate.

In one embodiment, the ST2 antigen binding protein is an antibodyanalog, sometimes referred to as “synthetic antibodies.” For example, avariety of work utilizes either alternative protein scaffolds orartificial scaffolds with grafted CDRs. Such scaffolds include, but arenot limited to, mutations introduced to stabilize the three-dimensionalstructure of the binding protein as well as wholly synthetic scaffoldsconsisting for example of biocompatible polymers. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as work based on antibody mimeticsutilizing fibronection components as a scaffold.

By “protein,” as used herein, is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells,as outlined below. Alternatively, the protein may include syntheticamino acids (e.g., homophenylalanine, citrulline, ornithine, andnorleucine), or peptidomimetic structures, i.e., “peptide or proteinanalogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad.Sci. U.S.A. 89:9367, incorporated by reference herein), which can beresistant to proteases or other physiological and/or storage conditions.Such synthetic amino acids may be incorporated in particular when theantigen binding protein is synthesized in vitro by conventional methodswell known in the art. In addition, any combination of peptidomimetic,synthetic and naturally occurring residues/structures can be used.“Amino acid” also includes imino acid residues such as proline andhydroxyproline. The amino acid “R group” or “side chain” may be ineither the (L)- or the (S)-configuration. In a specific embodiment, theamino acids are in the (L)- or (S)-configuration.

In certain aspects, the invention provides recombinant antigen bindingproteins that bind ST2 and, in some embodiments, a recombinant human ST2or portion thereof. In this context, a “recombinant protein” is aprotein made using recombinant techniques using any techniques andmethods known in the art, i.e., through the expression of a recombinantnucleic acid as described herein. Methods and techniques for theproduction of recombinant proteins are well known in the art.Embodiments of the invention include recombinant antigen bindingproteins that bind wild-type ST2 and variants thereof.

“Consisting essentially of” means that the amino acid sequence can varyby about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% relativeto the recited SEQ ID NO: sequence and still retain biological activity,as described herein.

In some embodiments, the antigen binding proteins of the invention areisolated proteins or substantially pure proteins. An “isolated” proteinis unaccompanied by at least some of the material with which it isnormally associated in its natural state, for example constituting atleast about 5%, or at least about 50% by weight of the total protein ina given sample. It is understood that the isolated protein mayconstitute from 5 to 99.9% by weight of the total protein contentdepending on the circumstances. For example, the protein may be made ata significantly higher concentration through the use of an induciblepromoter or high expression promoter, such that the protein is made atincreased concentration levels. The definition includes the productionof an antigen binding protein in a wide variety of organisms and/or hostcells that are known in the art.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs are at least 80% to the sequences depictedherein, and more typically with preferably increasing homologies oridentities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, and almost 100%. In a similar manner, “percent (%) nucleic acidsequence identity” with respect to the nucleic acid sequence of thebinding proteins identified herein is defined as the percentage ofnucleotide residues in a candidate sequence that are identical with thenucleotide residues in the coding sequence of the antigen bindingprotein. A specific method utilizes the BLASTN module of WU-BLAST-2 setto the default parameters, with overlap span and overlap fraction set to1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andthe nucleotide sequences depicted herein are at least 80%, and moretypically with preferably increasing homologies or identities of atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.

Thus, a “variant CDR” is one with the specified homology, similarity, oridentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the specificity and/or activity of the parent CDR.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of antigen binding protein activities, such as ST2 binding.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions may betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as TABLE 3.

TABLE 3 Original Exemplary Residue Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTABLE 3. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the antigen binding protein proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the antigen binding protein is altered. For example,glycosylation sites may be altered or removed as discussed herein.

Other derivatives of ST2 antibodies within the scope of this inventioninclude covalent or aggregative conjugates of ST2 antibodies, orfragments thereof, with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of a ST2 antibodypolypeptide. For example, the conjugated peptide may be a heterologoussignal (or leader) polypeptide, e.g., the yeast alpha-factor leader, ora peptide such as an epitope tag. ST2 antibody-containing fusionproteins can comprise peptides added to facilitate purification oridentification of the ST2 antibody (e.g., poly-His). A ST2 antibodypolypeptide also can be linked to the FLAG peptide as described in Hoppet al., Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912. TheFLAG peptide is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody (mAb), enabling rapid assay andfacile purification of expressed recombinant protein. Reagents usefulfor preparing fusion proteins in which the FLAG peptide is fused to agiven polypeptide are commercially available (Sigma, St. Louis, Mo.).

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a ST2 binding fragmentof a ST2 antibody to the Fc region of an antibody. The dimer can be madeby, for example, inserting a gene fusion encoding the fusion proteininto an appropriate expression vector, expressing the gene fusion inhost cells transformed with the recombinant expression vector, andallowing the expressed fusion protein to assemble much like antibodymolecules, whereupon interchain disulfide bonds form between the Fcmoieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of a ST2 antibody may be substituted for the variable portion ofan antibody heavy and/or light chain.

Another method for preparing oligomeric ST2 antibody derivativesinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising ST2 antibody fragmentor derivative fused to a leucine zipper peptide are expressed insuitable host cells, and the soluble oligomeric ST2 antibody fragmentsor derivatives that form are recovered from the culture supernatant.

Covalent modifications of antigen binding proteins are included withinthe scope of this invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antigen binding protein are introduced into themolecule by reacting specific amino acid residues of the antigen bindingprotein with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantigen binding proteins to a water-insoluble support matrix or surfacefor use in a variety of methods. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the antigen binding proteinincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the target polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antigen binding proteincomprises linking the antigen binding protein to variousnonproteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, asis known in the art, amino acid substitutions may be made in variouspositions within the antigen binding protein to facilitate the additionof polymers such as PEG.

In some embodiments, the covalent modification of the antigen bindingproteins of the invention comprises the addition of one or more labels.

The term “labeling group” means any detectable label. Examples ofsuitable labeling groups include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labeling group is coupled to the antigenbinding protein via spacer arms of various lengths to reduce potentialsteric hindrance. Various methods for labeling proteins are known in theart and may be used in performing the present invention.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in performing thepresent invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, CASCADE BLUEJ dye,TEXAS RED dye, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LCRed 705, OREGON GREEN dye, the ALEXA FLUOR dyes (ALEXA FLUOR 350 dye,ALEXA FLUOR 430 dye, ALEXA FLUOR 488 dye, ALEXA FLUOR 546 dye, ALEXAFLUOR 568 dye, ALEXA FLUOR 594 dye, ALEXA FLUOR 633 dye, ALEXA FLUOR 660dye, ALEXA FLUOR 680 dye), CASCADE BLUE dye, CASCADE YELLOW dye andR-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhodamine,and TEXAS RED dye (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (AmershamLife Science, Pittsburgh, Pa.). Suitable optical dyes, includingfluorophores, are described in Molecular Probes Handbook by Richard P.Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

The exemplary antigen binding proteins described herein have propertiesbased on the distinct epitope on ST2 bound by the antigen bindingprotein. The term “epitope” means the amino acids of a target moleculethat are contacted by an antigen binding protein, e.g., an antibody,when the antigen binding protein is bound to the target molecule. Anepitope can be contiguous or non-contiguous (e.g., (i) in a single-chainpolypeptide, amino acid residues that are not contiguous to one anotherin the polypeptide sequence but that within the context of the targetmolecule are bound by the antigen binding protein, or (ii) in amultimeric receptor comprising two or more individual components, e.g.,ST2 and AcP, amino acid residues are present on one or more of theindividual components but are still bound by the antigen bindingprotein. Epitope determinants can include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl or sulfonyl groups, and can have specific three dimensionalstructural characteristics, and/or specific charge characteristics.Generally, antigen binding proteins specific for a particular targetmolecule will preferentially recognize an epitope on the target moleculein a complex mixture of proteins and/or macromolecules.

Methods of characterizing the epitope bound by an antigen bindingprotein are well known in the art, including, but not limited to,binning (cross-competition) (Miller et al “Epitope binning of murinemonoclonal antibodies by a multiplexed pairing assay” J Immunol Methods(2011) 365, 118-25), peptide mapping (e.g., PEPSPOT™) (Albert et al “TheB-cell Epitope of the Monoclonal Anti-Factor VIII Antibody ESH8Characterized by Peptide Array Analysis” 2008 Thromb Haemost 99, 634-7),mutagenesis methods such as chimeras (Song et al “Epitope Mapping ofIbalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1Activity in Infected Patients” J. Virol. (2010) 84, 6935-6942), alaninescanning (Cunningham and Wells “High-resolution epitope mapping ofHGH-receptor interactions by alanine-scanning mutagenesis” Science(1989) 244, 1081-1085), arginine scanning (Lim et al “A diversity ofantibody epitopes can induce signaling through the erythropoietinreceptor” Biochemistry (2010) 49, 3797-3804), HD exchange methods(Coates et al “Epitope mapping by amide hydrogen/deuterium exchangecoupled with immobilization of antibody, on-line proteolysis, liquidchromatography and mass spectrometry” Rapid Commun. Mass Spectrom.(2009) 23 639-647), NMR cross saturation methods (Morgan et al “Preciseepitope mapping of malaria parasite inhibitory antibodies by TROSY NMRcross-saturation” Biochemistry (2005) 44, 518-23), and crystallography(Gerhardt et al “Structure of IL-17A in complex with a potent, fullyhuman neutralizing antibody” J. Mol. Biol (2009) 394, 905-21). Themethods vary in the level of detail they provide as to the amino acidscomprising the epitope.

Antigen binding proteins of the present invention include those thathave an overlapping epitope with an exemplary antigen binding proteindescribed herein, e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab30, Ab32, or Ab33. In certain embodiments, the antigenbinding protein has an identical epitope as to the exemplary antigenbinding proteins. In other embodiments, the antigen binding proteinbinds only a subset of the same amino acids as the exemplary antigenbinding protein.

In certain embodiments, the ST2 antigen binding protein has an identicalor overlapping epitope as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab30, Ab32, or Ab33 and comprises a) a light chain variabledomain having at least 90% identity, at least 95% identity, or isidentical to the amino acid sequence set forth in SEQ ID NO:95, SEQ IDNO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQID NO:163, SEQ ID NO:164, or SEQ ID NO:165; b) a heavy chain variabledomain having at least 90% identity, at least 95% identity, or isidentical to the amino acid sequence set forth in SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:145, SEQ ID NO:146, or SEQ ID NO:147; or c) the light chain variabledomain of a) and the heavy chain variable domain of b).

In certain embodiments, the ST2 antigen binding protein has an identicalor overlapping epitope as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab30, Ab32, or Ab33, and comprises a light chain variabledomain having at least 90%, at least 95%, or is identical to the aminoacid sequence set forth in SEQ ID NO:95 and a heavy chain variabledomain having at least 90%, at least 95%, or is identical to the aminoacid sequence set forth in SEQ ID NO:29; those comprising a light chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:96 and a heavy chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:30; those comprising alight chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:97 and aheavy chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:31; thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:98 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:32; those comprising a light chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:99 and a heavy chain variable domain having at least 90%,at least 95%, or is identical to the amino acid sequence set forth inSEQ ID NO:33; those comprising a light chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:100 and a heavy chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:34; those comprising a light chain variable domain havingat least 90%, at least 95%, or is identical to the amino acid sequenceset forth in SEQ ID NO:101 and a heavy chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:35; those comprising a light chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:102 and a heavy chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:36; those comprising a light chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:103 and a heavy chainvariable domain having at least 90%, at least 95%, or is identical tothe amino acid sequence set forth in SEQ ID NO:37; those comprising alight chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:104 and aheavy chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:38; thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:105 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:39; those comprising a light chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:163 and a heavy chain variable domain having at least 90%,at least 95%, or is identical to the amino acid sequence set forth inSEQ ID NO:145; those comprising a light chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:164 and a heavy chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:146; and those comprising a light chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:165 and a heavy chain variable domainhaving at least 90%, at least 95%, or is identical to the amino acidsequence set forth in SEQ ID NO:147.

In certain embodiments, the ST2 antigen binding protein has an identicalor overlapping epitope as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab30, Ab32, or Ab33 and comprises a) a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQID NO:104, SEQ ID NO:105, SEQ ID NO:163, SEQ ID NO:164, or SEQ IDNO:165; b) a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:145, SEQ IDNO:146, or SEQ ID NO:147; or c) the light chain variable domain of a)and the heavy chain variable domain of b).

In certain embodiments, the ST2 antigen binding protein has an identicalor overlapping epitope as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab30, Ab32, or Ab33 and comprises a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:95 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:29;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:96 and a heavy chainvariable domain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:30; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:97 and a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:31; those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:98 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:32;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:99 and a heavy chainvariable domain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:33; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:100 and a heavy chain variable domain having no more than ten orno more than five amino acid additions, deletions or substitutions fromthe amino acid sequence set forth in SEQ ID NO:34; those comprising alight chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:101 and a heavy chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:35; those comprising a light chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:102and a heavy chain variable domain having no more than ten or no morethan five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:36; those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:103 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:37;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:104 and a heavychain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:38; those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:105 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:39;those comprising a light chain variable domain having no more than tenor no more than five amino acid additions, deletions or substitutionsfrom the amino acid sequence set forth in SEQ ID NO:163 and a heavychain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:145; those comprising a light chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:164 and a heavy chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:146;and those comprising a light chain variable domain having no more thanten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:165and a heavy chain variable domain having no more than ten or no morethan five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:147.

In certain embodiments, the ST2 antigen binding protein has an identicalor overlapping epitope as Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab30, Ab32, or Ab33 and comprises a light chain variabledomain comprising a) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:106; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:117; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:128; b) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:107; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:118; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:129; c) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:108; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:119; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:130; d) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:109; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:120; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:131; e) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:110; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:121; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:132; f) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:111; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:122; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:133; g) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:112; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:123; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:134; h) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:113; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:124; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:135; i) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:114; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:125; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:136; j) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:115; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:126; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:137; k) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:116; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:127; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:138; l) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:166; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:169; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:172; m) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:167; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:170; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:173; or n) an LCDR1 having no more than three amino acidadditions, deletions, or substitutions from the LCDR1 sequence set forthin SEQ ID NO:168; an LCDR2 having no more than three amino acidadditions, deletions, or substitutions from the LCDR2 sequence set forthin SEQ ID NO:171; and an LCDR3 having no more than three amino acidadditions, deletions, or substitutions from the LCDR3 sequence set forthin SEQ ID NO:174; and a heavy chain variable domain comprising o) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:40; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:51; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:62; p) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:41; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:52; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:63; q) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:42; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:53; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:64; r) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:43; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:54; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:65; s) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:44; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:55; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:66; t) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:45; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:56; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:67; u) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:46; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:57; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:68; v) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:47; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:58; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:69; w) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:48; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:59; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:70; x) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:49; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:60; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:71; y) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:50; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:61; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:72; z) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:148; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:151; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:154; aa) anHCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:149; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:152; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:155; or bb)an HCDR1 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR1 sequence set forth in SEQ ID NO:150; anHCDR2 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR2 sequence set forth in SEQ ID NO:153; and anHCDR3 having no more than three amino acid additions, deletions, orsubstitutions from the HCDR3 sequence set forth in SEQ ID NO:156.

Preferred ST2 antigen binding proteins described immediately aboveinclude those comprising the light chain variable domain of a) and theheavy chain variable domain of o); those comprising the light chainvariable domain of b) and the heavy chain variable domain of p); thosecomprising the light chain variable domain of c) and the heavy chainvariable domain of q); those comprising the light chain variable domainof d) and the heavy chain variable domain of r); those comprising thelight chain variable domain of e) and the heavy chain variable domain ofs); those comprising the light chain variable domain of f) and the heavychain variable domain of t); those comprising the light chain variabledomain of g) and the heavy chain variable domain of u); those comprisingthe light chain variable domain of h) and the heavy chain variabledomain of v); those comprising the light chain variable domain of i) andthe heavy chain variable domain of w); those comprising the light chainvariable domain of j) and the heavy chain variable domain of x); thosecomprising the light chain variable domain of k) and the heavy chainvariable domain of y); those comprising the light chain variable domainof l) and the heavy chain variable domain of z); those comprising thelight chain variable domain of m) and the heavy chain variable domain ofaa); and those comprising the light chain variable domain of n) and theheavy chain variable domain of bb).

Antigen binding proteins that have an identical epitope or overlappingepitope will often cross-compete for binding to the antigen. Thus, incertain embodiments, an antigen binding protein of the inventioncross-competes with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10,Ab11, Ab30, Ab32, or Ab33. To “cross-compete” or “cross-competition”means the antigen binding proteins compete for the same epitope orbinding site on a target. Such competition can be determined by an assayin which the reference antigen binding protein (e.g., antibody orantigen-binding portion thereof) prevents or inhibits specific bindingof a test antigen binding protein, and vice versa. Numerous types ofcompetitive binding assays can be used to determine if a test moleculecompetes with a reference molecule for binding. Examples of assays thatcan be employed include solid phase direct or indirect radioimmunoassay(RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see, e.g., Stahli et al. (1983) Methods in Enzymology9:242-253), solid phase direct biotin-avidin EIA (see, e.g., Kirkland etal., (1986) J. Immunol. 137:3614-3619), solid phase direct labeledassay, solid phase direct labeled sandwich assay, Luminex (Jia et al “Anovel method of Multiplexed Competitive Antibody Binning for thecharacterization of monoclonal antibodies” J. Immunological Methods(2004) 288, 91-98) and surface plasmon resonance ((Song et al “EpitopeMapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody withAnti-HIV-1 Activity in Infected Patients” J. Virol. (2010) 84,6935-6942). An exemplary method of determining cross-competition isdescribed in Example 5. Usually, when a competing antigen bindingprotein is present in excess, it will inhibit binding of a referenceantigen binding protein to a common antigen by at least 50%, 55%, 60%,65%, 70%, or 75%. In some instances, binding is inhibited by at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

Ab2 binds human and cyo ST2 with high affinity and blocks IL-33 bindingto ST2, thus blocking IL-33 mediated ST2 signalling. Antibodies withidentical to, similar, or overlapping epitopes with Ab2 may share theseunique characteristics. In preferred embodiments, an ST2 antigen bindingprotein cross-competes with Ab2 for binding to ST2. Exemplary ST2antigen binding proteins that cross-compete with Ab2 include Ab1, Ab3,Ab5, Ab7, Ab8, and Ab30 (see Example 5). If attempting to findantibodies that bind an overlapping, similar, or identical eptiope asAb2, one may screen one or more antibodies for cross-competition withAb2. Moreover, when making variants to an antibody that cross-reactswith Ab2, one may screen such antibodies to determine if thecross-competition is maintained after variation, suggesting that theepitope of the variant is not significantly altered from the parentmolecule. Thus, in certain embodiments, the invention provides antibodyvariants that cross-compete with Ab2 for binding to ST2.

Besides cross-competing with each other, antibodies with overlapping,similar, or identical epitopes may be affected by mutagenesis of ST2similarly. Certain mutations may inhibit binding of an antibody; othersmay enhance or activate binding. In Example 11, scanningarginine/alanine mutagenesis was performed on a portion of theextracellular domain of ST2 and the effect on exemplary antibodiesdetermined. Included with the scope of the invention are ST2 bindingproteins having characteristics such that they are affected in a similarway as an exemplary antibody to mutagenesis.

In certain embodiments, binding of an ST2 antigen binding protein isinhibited by a single mutation in ST2, wherein the single mutation isselected from the group consisting of L14R, I15R, S33R, E43R, V47R,A62R, G65R, T79R, D92R, D97R, V104R, G138R, N152R, and V176R. Inpreferred embodiments, any of two or more, three or more, four or more,five or more, six or more, seven or more, eight or more nine or more,ten or more, or all of the single mutations of the group individuallyinhibit binding of the ST2 binding protein. In other embodiments,binding of an ST2 antigen binding protein is activated by a singlemutation in ST2, wherein the single mutation is selected from the groupconsisting of L53R, R72A, and S73R. In preferred embodiments, all of thesingle mutations of the group individually activate binding of the ST2binding protein. In preferred embodiments, the ST2 antigen bindingprotein shares the attributes of Ab2 and are inhibited by any of L14R,I15R, S33R, E43R, V47R, A62R, G65R, T79R, D92R, D97R, V104R, G138R,N152R, and V176R and are activated by any of L53R, R72A, S73R.

Another method of characterizing an antibody based on its epitope isamide hydrogen/deuterium exchange (HDX). HDX has been widely used tostudy protein conformation and dynamics, protein-ligand interactions andprotein-protein interactions (Zhang and Smith 1993, Engen and Smith2001). Mass spectrometric detection provides a powerful tool todetermine the extent of the exchange, because the replacement of asingle hydrogen with deuterium results in a mass increase of 1 Da foreach exchange. The extent of HDX can be readily measured at the peptidelevel by analysis of the protein proteolytic digest by liquidchromatography in conjunction with tandem mass spectrometry undercontrolled conditions (Engen and Smith 2001, Baerga-Ortiz, Hughes et al.2002. Codreanu, Ladner et al. 2002. Hamuro, Coales et al. 2006, Coales,Tuske et al. 2009, Zhang, Zhang et al. 2012).

Comparison of antigen HDX profiles between proteotlyic digests of ST2with the absence and the presence of an antibody (free versus boundstate) can reveal the interaction sites. Specifically, when the antibodybinds to ST2, solvent accessible amide hydrogens in free ST2 can becomeprotected, and as a result, slower exchange rates are observed.Therefore, regions that gained less deuterium in the presence of theantibody than in its absence are potential binding epitopes. Otherfactors, including exchange rate in the free-state, knowledge of theantigen protein structure, as well as results from other epitope mappingefforts, are considered when the epitopes are determined.

Ab2 binding to ST2 was analyzed by HDX as described in Example 12. Theanalysis demonstrated that Ab2 binds to/alters the exchange rate of theportion of the ST2 structure comprising amino acids 33-44 and 88-94 ofamino acids 19-322 of SEQ ID NO:1 (amino acids 15-26 and 70-76 of matureST2, respectively). Antibodies with overlapping epitopes, similar, oridentical epitopes as Ab2 will also bind to/alter the exchange rate ofamino acids within 33-44 and 88-94 of SEQ ID NO:1. In certainembodiments, an ST2 binding protein, e.g., antibody, protects any of theamino acids 33-44 of SEQ ID NO:1 when bound to ST2 and analyzed by HDX.In other embodiments, any of amino acids 88-94 are protected. Bothindicate partial overlap of binding epitopes with Ab2. In preferredembodiments, both any of 33-44 and any of 88-94 are protected. Incertain embodiments, an ST2 binding protein, e.g., antibody, protectsall of the amino acids 33-44 of SEQ ID NO:1 when bound to ST2 andanalyzed by HDX. In other embodiments, all of amino acids 88-94 areprotected. Both indicate a similar binding epitope with Ab2. Inpreferred embodiments, both all of 33-44 and all of 88-94 are protected,indicating an identical or nearly identical epitope as Ab2.

Binding of Ab2 to ST2 was further analyzed using X-ray crystallography.The X-ray crystallography was consistent with the HDX analysis. Theinterface between the Ab and the antigen can be determined/defined anumber of ways. In Example 13, the interface was determined usingsolvent exposure differential and by distance. ST2 residues that arewithin the interface with Ab2 as determined by solvent exposuredifferences or distance of less than 5A are (corresponding to positionin mature ST2 (lacking leader sequence)) K1, F2, P19, R20, Q21, G22,K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79, and Y81. Incertain embodiments, the ST2 binding protein forms an interface with ST2that overlaps with that of Ab2, including those wherein any of K1, F2,P19, R20, Q21, G22, K23, Y26, I70, V71, R72, S73, P74, T75, F76, N77,R78, T79, or Y81 are within the interface. In some embodiments, the ST2binding protein forms an interface with ST2 wherein P19, R20, Q21, G22,K23, and/or Y26 are within the interface. In other embodiments, I70,V71, R72, S73, P74, T75, F76, N77, R78, T79, and/or Y81 are within theinterface. In preferred embodiments, K1, F2, P19, R20, Q21, G22, K23,Y26, I70, V71, R72, S73, P74, T75, F76, N77, R78, T79, and Y81 arewithin the interface.

The crystal structure indicated that certain amino acid residues formedhydrogen bonds or salt bridges with amino acids with Ab2. Those residuesinclude K1, R20, K23, Y26, T75, N77, R78, and T79. In certainembodiments, an ST2 antigen binding protein forms hydrogen bonds or asalt bridge with one or more of K1, R20, K23, Y26, T75, N77, R78, andT79.

The crystal structure further provides information as to which residuesof Ab2 form the interface with ST2. FIG. 10 indicates the residues inthe light chain variable region and heavy chain variable region thatform an interface with ST2. Also indicated are the residues that formhydrogen bonds or salt bridges with amino acids in ST2. One may use thisinformation to design variants of Ab2, including those that containvariable domains having 90% identity, 95% identity, and 10 or lessinsertions, deletions, and/or substitutions within the light chain orheavy chain variable domain of Ab2. One may wish to maintain the aminoacids within the interface while altering non-interface residues. Thus,one may design and create variants of Ab2 having one or more amino acidadditions, substitutions, and/or deletions within one or more CDRs ofAb2 that maintain binding to ST2.

In some embodiments, an ST2 binding protein comprises a variant of Ab2light chain variable region (SEQ ID NO:96) wherein D28, I29, S30, N31,Y32, Y49, D50, N53, E55, T56, D91, D92, N93, F94, and/or L96 remainunchanged or comprise a conservative substitution thereof, and/or avariant of Ab2 heavy chain variable region (SEQ ID NO:30) wherein W33,I50, D57, R59, H99, G100, T101, S102, S103, D104, Y105, and/or Y106remain unchanged or comprise a conservative mutation. In preferredembodiments, D28, N31, D50, N53, E55, D91 and D92 of the light chainvariable region remain unchanged and S102, S103, D104, and Y105 of theheavy chain remain unchanged.

Polynucleotides Encoding ST2 Antigen Binding Proteins

Encompassed within the invention are nucleic acids encoding ST2 antigenbinding proteins, including antibodies, as defined herein. Preferrednucleic acids include those that encode the exemplary light and heavychains described herein.

An exemplary nucleic acid encoding Ab1 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:73.

An exemplary nucleic acid encoding Ab2 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:74.

An exemplary nucleic acid encoding Ab3 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:75.

An exemplary nucleic acid encoding Ab4 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:76.

An exemplary nucleic acid encoding Ab5 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:77.

An exemplary nucleic acid encoding Ab6 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:78.

An exemplary nucleic acid encoding Ab7 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:79.

An exemplary nucleic acid encoding Ab8 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:80.

An exemplary nucleic acid encoding Ab9 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:81.

An exemplary nucleic acid encoding Ab10 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:82.

An exemplary nucleic acid encoding Ab11 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:83.

An exemplary nucleic acid encoding Ab30 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:157.

An exemplary nucleic acid encoding Ab32 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:158.

An exemplary nucleic acid encoding Ab33 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:159.

An exemplary nucleic acid encoding Ab1 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:7.

An exemplary nucleic acid encoding Ab2 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:8.

An exemplary nucleic acid encoding Ab3 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:9.

An exemplary nucleic acid encoding Ab4 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:10.

An exemplary nucleic acid encoding Ab5 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:11.

An exemplary nucleic acid encoding Ab6 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:12.

An exemplary nucleic acid encoding Ab7 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:13.

An exemplary nucleic acid encoding Ab8 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:14.

An exemplary nucleic acid encoding Ab9 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:15.

An exemplary nucleic acid encoding Ab10 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:16.

An exemplary nucleic acid encoding Ab11 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:17.

An exemplary nucleic acid encoding Ab30 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:139.

An exemplary nucleic acid encoding Ab32 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:140.

An exemplary nucleic acid encoding Ab33 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:141.

Aspects of the invention include polynucleotide variants (e.g., due todegeneracy) that encode the amino acid sequences described herein.

Aspects of the invention include a variety of embodiments including, butnot limited to, the following exemplary embodiments.

An isolated polynucleotide, wherein said polynucleotide encodes one ormore polypeptides comprising an amino acid sequence selected from thegroup consisting of:

A. 1. a light chain variable domain sequence that is at least 90%identical to a light chain variable domain sequence set forth in SEQ IDNOs:95-105, 163-165;

-   -   2. a heavy chain variable domain sequence that is at least 90%        identical to a heavy chain variable domain sequence set forth in        SEQ ID NOs:29-39, 145-147;    -   3. a light chain variable domain of (1) and a heavy chain        variable domain of (2); and

B. a light chain variable domain comprising a CDR1, CDR2, CDR3 and/or aheavy chain variable domain comprising a CDR1, CDR2, CDR3 that are thesame or differ by no more than a total of three amino acid additions,substitutions, and/or deletions in each CDR from the followingsequences:

-   -   1. a light chain CDR1 (SEQ ID NO:106), CDR2 (SEQ ID NO:117),        CDR3 (SEQ ID NO:128) or a heavy chain CDR1 (SEQ ID NO:40), CDR2        (SEQ ID NO:51), CDR3 (SEQ ID NO:62) of Ab1;    -   2. a light chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:118),        CDR3 (SEQ ID NO:129) or a heavy chain CDR1 (SEQ ID NO:41), CDR2        (SEQ ID NO:52), CDR3 (SEQ ID NO:63) of Ab2;    -   3. a light chain CDR1 (SEQ ID NO:108), CDR2 (SEQ ID NO:119),        CDR3 (SEQ ID NO:130) or a heavy chain CDR1 (SEQ ID NO:42), CDR2        (SEQ ID NO:53), CDR3 (SEQ ID NO:64) of Ab3;    -   4. a light chain CDR1 (SEQ ID NO:109), CDR2 (SEQ ID NO:120),        CDR3 (SEQ ID NO:131) or a heavy chain CDR1 (SEQ ID NO:43), CDR2        (SEQ ID NO:54), CDR3 (SEQ ID NO:65) of Ab4;    -   5. a light chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:121),        CDR3 (SEQ ID NO:132) or a heavy chain CDR1 (SEQ ID NO:44), CDR2        (SEQ ID NO:55), CDR3 (SEQ ID NO:66) of Ab5;    -   6. a light chain CDR1 (SEQ ID NO:111), CDR2 (SEQ ID NO:122),        CDR3 (SEQ ID NO:133) or a heavy chain CDR1 (SEQ ID NO:45), CDR2        (SEQ ID NO:56), CDR3 (SEQ ID NO:67) of Ab6;    -   7. a light chain CDR1 (SEQ ID NO:112), CDR2 (SEQ ID NO:123),        CDR3 (SEQ ID NO:134) or a heavy chain CDR1 (SEQ ID NO:46), CDR2        (SEQ ID NO:57), CDR3 (SEQ ID NO:68) of Ab7;    -   8. a light chain CDR1 (SEQ ID NO:113), CDR2 (SEQ ID NO:124),        CDR3 (SEQ ID NO:135) or a heavy chain CDR1 (SEQ ID NO:47), CDR2        (SEQ ID NO:58), CDR3 (SEQ ID NO:69) of Ab8;    -   9. a light chain CDR1 (SEQ ID NO:114), CDR2 (SEQ ID NO:125),        CDR3 (SEQ ID NO:136) or a heavy chain CDR1 (SEQ ID NO:48), CDR2        (SEQ ID NO:59), CDR3 (SEQ ID NO:70) of Ab9;    -   10. a light chain CDR1 (SEQ ID NO:115), CDR2 (SEQ ID NO:126),        CDR3 (SEQ ID NO:137) or a heavy chain CDR1 (SEQ ID NO:49), CDR2        (SEQ ID NO:60), CDR3 (SEQ ID NO:71) of Ab10;    -   11. a light chain CDR1 (SEQ ID NO:116), CDR2 (SEQ ID NO:127),        CDR3 (SEQ ID NO:138) or a heavy chain CDR1 (SEQ ID NO:50), CDR2        (SEQ ID NO:61), CDR3 (SEQ ID NO:72) of Ab11;    -   12. a light chain CDR1 (SEQ ID NO:166), CDR2 (SEQ ID NO:169),        CDR3 (SEQ ID NO:172) or a heavy chain CDR1 (SEQ ID NO:148), CDR2        (SEQ ID NO:151), CDR3 (SEQ ID NO:154) of Ab30;    -   13. a light chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:170),        CDR3 (SEQ ID NO:173) or a heavy chain CDR1 (SEQ ID NO:149), CDR2        (SEQ ID NO:152), CDR3 (SEQ ID NO:155) of Ab32; and    -   14. a light chain CDR1 (SEQ ID NO:168), CDR2 (SEQ ID NO:171),        CDR3 (SEQ ID NO:174) or a heavy chain CDR1 (SEQ ID NO:150), CDR2        (SEQ ID NO:153), CDR3 (SEQ ID NO:156) of Ab33.

In preferred embodiments, the polypeptide encoded by the isolatednucleic acid is a component of an antigen binding protein that bindsST2.

Nucleotide sequences corresponding to the amino acid sequences describedherein, to be used as probes or primers for the isolation of nucleicacids or as query sequences for database searches, can be obtained by“back-translation” from the amino acid sequences, or by identificationof regions of amino acid identity with polypeptides for which the codingDNA sequence has been identified. The well-known polymerase chainreaction (PCR) procedure can be employed to isolate and amplify a DNAsequence encoding a ST2 antigen binding proteins or a desiredcombination of ST2 antigen binding protein polypeptide fragments.Oligonucleotides that define the desired termini of the combination ofDNA fragments are employed as 5′ and 3′ primers. The oligonucleotidescan additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified combination ofDNA fragments into an expression vector. PCR techniques are described inSaiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu etal., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCRProtocols: A Guide to Methods and Applications, Innis et. al., eds.,Academic Press, Inc. (1990).

Nucleic acid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. The nucleic acid molecules of the invention include full-lengthgenes or cDNA molecules as well as a combination of fragments thereof.The nucleic acids of the invention are preferentially derived from humansources, but the invention includes those derived from non-humanspecies, as well.

An “isolated nucleic acid” is a nucleic acid that has been separatedfrom adjacent genetic sequences present in the genome of the organismfrom which the nucleic acid was isolated, in the case of nucleic acidsisolated from naturally-occurring sources. In the case of nucleic acidssynthesized enzymatically from a template or chemically, such as PCRproducts, cDNA molecules, or oligonucleotides for example, it isunderstood that the nucleic acids resulting from such processes areisolated nucleic acids. An isolated nucleic acid molecule refers to anucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

The present invention also includes nucleic acids that hybridize undermoderately stringent conditions, and more preferably highly stringentconditions, to nucleic acids encoding ST2 antigen binding proteins asdescribed herein. The basic parameters affecting the choice ofhybridization conditions and guidance for devising suitable conditionsare set forth by Sambrook, Fritsch, and Maniatis (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11; and Current Protocols inMolecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4), and can be readily determined by thosehaving ordinary skill in the art based on, for example, the lengthand/or base composition of the DNA. One way of achieving moderatelystringent conditions involves the use of a prewashing solutioncontaining 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization bufferof about 50% formamide, 6×SSC, and a hybridization temperature of about55 degrees C. (or other similar hybridization solutions, such as onecontaining about 50% formamide, with a hybridization temperature ofabout 42 degrees C.), and washing conditions of about 60 degrees C., in0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined ashybridization conditions as above, but with washing at approximately 68degrees C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mMNaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC(1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization andwash buffers; washes are performed for 15 minutes after hybridization iscomplete. It should be understood that the wash temperature and washsalt concentration can be adjusted as necessary to achieve a desireddegree of stringency by applying the basic principles that governhybridization reactions and duplex stability, as known to those skilledin the art and described further below (see, e.g., Sambrook et al.,1989). When hybridizing a nucleic acid to a target nucleic acid ofunknown sequence, the hybrid length is assumed to be that of thehybridizing nucleic acid. When nucleic acids of known sequence arehybridized, the hybrid length can be determined by aligning thesequences of the nucleic acids and identifying the region or regions ofoptimal sequence complementarity. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be 5to 10.degrees C. less than the melting temperature (Tm) of the hybrid,where Tm is determined according to the following equations. For hybridsless than 18 base pairs in length, Tm (degrees C.)=2(# of A+T bases)+4(#of #G+C bases). For hybrids above 18 base pairs in length, Tm (degreesC.)=81.5+16.6(log₁₀ [Na])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165M). Preferably, each suchhybridizing nucleic acid has a length that is at least 15 nucleotides(or more preferably at least 18 nucleotides, or at least 20 nucleotides,or at least 25 nucleotides, or at least 30 nucleotides, or at least 40nucleotides, or most preferably at least 50 nucleotides), or at least25% (more preferably at least 50%, or at least 60%, or at least 70%, andmost preferably at least 80%) of the length of the nucleic acid of thepresent invention to which it hybridizes, and has at least 60% sequenceidentity (more preferably at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, and mostpreferably at least 99.5%) with the nucleic acid of the presentinvention to which it hybridizes, where sequence identity is determinedby comparing the sequences of the hybridizing nucleic acids when alignedso as to maximize overlap and identity while minimizing sequence gaps asdescribed in more detail above.

The variants according to the invention are ordinarily prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the antigenbinding protein, using cassette or PCR mutagenesis or other techniqueswell known in the art, to produce DNA encoding the variant, andthereafter expressing the recombinant DNA in cell culture as outlinedherein. However, antigen binding protein fragments comprising variantCDRs having up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, e.g., binding to ST2, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the CDRs (and heavy and light chains or othercomponents of the antigen binding protein) of the present invention.Thus, having identified a particular amino acid sequence, those skilledin the art could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the encoded protein.

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the ST2 antigenbinding protein coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the ST2 antigen binding protein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified ST2 antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thyrnidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein antibody that binds to ST2 polypeptide. As aresult, increased quantities of a polypeptide such as an ST2 antigenbinding protein are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed. In certain embodiments, one or more coding regions may beoperably linked to an internal ribosome binding site (IRES), allowingtranslation of two open reading frames from a single RNA transcript.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the ST2 antigen binding protein. Promoters areuntranscribed sequences located upstream (i.e., 5′) to the start codonof a structural gene (generally within about 100 to 1000 bp) thatcontrol transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an ST2 antigen binding protein of theinvention by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence into thevector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising aST2 antigen binding protein of the invention by higher eukaryotes.Enhancers are cis-acting elements of DNA, usually about 10-300 bp inlength, that act on the promoter to increase transcription. Enhancersare relatively orientation and position independent, having been foundat positions both 5′ and 3′ to the transcription unit. Several enhancersequences available from mammalian genes are known (e.g., globin,elastase, albumin, alpha-feto-protein and insulin). Typically, however,an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The vector may contain one or more elements that facilitate expressionwhen the vector is integrated into the host cell genome. Examplesinclude an EASE element (Aldrich et al. 2003 Biotechnol Prog.19:1433-38) and a matrix attachment region (MAR). MARs mediatestructural organization of the chromatin and may insulate the integratedvector from “position” effect. Thus, MARs are particularly useful whenthe vector is used to create stable transfectants. A number of naturaland synthetic MAR-containing nucleic acids are known in the art, e.g.,U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974;7,259,010; 6,037,525; 7,422,874; 7,129,062.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an ST2 antigen binding sequence has been inserted into theproper site of the vector, the completed vector may be inserted into asuitable host cell for amplification and/or polypeptide expression. Thetransformation of an expression vector for an ST2 antigen bindingprotein into a selected host cell may be accomplished by well knownmethods including transfection, infection, calcium phosphateco-precipitation, electroporation, microinjection, lipofection,DEAE-dextran mediated transfection, or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anST2 antigen binding protein that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule. A host cell may be eukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC) and any celllines used in an expression system known in the art can be used to makethe recombinant polypeptides of the invention. In general, host cellsare transformed with a recombinant expression vector that comprises DNAencoding a desired anti-ST2 antibody polypeptide. Among the host cellsthat may be employed are prokaryotes, yeast or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include insect cellsand established cell lines of mammalian origin. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells,C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells,or their derivatives such as Veggie CHO and related cell lines whichgrow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cellline derived from the African green monkey kidney cell line CVI (ATCCCCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, humanembryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermalA431 cells, human Colo205 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49,for example, can be used for expression of the polypeptide when it isdesirable to use the polypeptide in various signal transduction orreporter assays. Alternatively, it is possible to produce thepolypeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Suitable yeasts include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous polypeptides. Suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous polypeptides. If the polypeptide is made in yeast orbacteria, it may be desirable to modify the polypeptide producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain the functional polypeptide. Suchcovalent attachments can be accomplished using known chemical orenzymatic methods. The polypeptide can also be produced by operablylinking the isolated nucleic acid of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and Methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987), andLuckow and Summers, Bio/Technology 6:47 (1988). Cell-free translationsystems could also be employed to produce polypeptides using RNAsderived from nucleic acid constructs disclosed herein. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, New York, 1985). A host cellthat comprises an isolated nucleic acid of the invention, preferablyoperably linked to at least one expression control sequence, is a“recombinant host cell”.

In certain embodiments, cell lines may be selected through determiningwhich cell lines have high expression levels and constitutively produceantigen binding proteins with ST2 binding properties. In anotherembodiment, a cell line from the B cell lineage that does not make itsown antibody but has a capacity to make and secrete a heterologousantibody can be selected.

Cell-Depletine ST2 Antigen Binding Proteins

In preferred embodiments, the ST2 antigen binding protein binds ST2 andinhibits IL-33 binding, thereby reducing IL-33 mediated signaling inST2-expressing cells. In certain embodiments, however, the ST2 antigenbinding protein binds ST2 and targets an ST2-expressing cell fordepletion. Of course, the ST2 antigen binding protein may inhibit IL-33binding and target the ST2 cell for depletion.

Cell-depleting ST2 antigen binding proteins are particularly useful fortreating diseases associated with over expression of ST2, e.g., aninflammatory disease or an ST2-expressing tumor. Methods of targetingcells with antigen binding proteins, e.g. antibodies, are well known inthe art. Exemplary embodiments are discussed below.

Antibody Drug Conjugates

Embodiments of the invention include antibody drug conjugates (ADCs).Generally the ADC comprises an antibody conjugated to a chemotherapeuticagent, e.g., a cytotoxic agent, a cytostatic agent, a toxin, or aradioactive agent. A linker molecule can be used to conjugate the drugto the antibody. A wide variety of linkers and drugs useful in ADCtechnology are known in the art and may be used in embodiments of thepresent invention. (See US20090028856; US2009/0274713; US2007/0031402;WO2005/084390; WO2009/099728; U.S. Pat. No. 5,208,020; U.S. Pat. No.5,416,064; U.S. Pat. Nos. 5,475,092; 5,585,499; 6,436,931; 6,372,738;and 6340701, all incorporated herein by reference).

Linkers

In certain embodiments, the ADC comprises a linker made up of one ormore linker components. Exemplary linker components include6-maleimidocaproyl, maleimidopropanoyl, valine-citrulline,alanine-phenylalanine, p-aminobenzyloxycarbonyl, and those resultingfrom conjugation with linker reagents, including, but not limited to,N-succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC,” also referred toherein also as “MCC”), and N-succinimidyl (4-iodo-acetyl)aminobenzoate(“SIAB”).

Linkers may be a “cleavable” linker or a “non-cleavable” linker (Ducryand Stump, Bioconjugate Chem. 2010, 21, 5-13; incorporated herein byreference in its entirety) Cleavable linkers are designed to release thedrug when subjected to certain environment factors, e.g., wheninternalized into the target cell. Cleavable linkers include acid labilelinkers, protease sensitive linkers, photolabile linkers, dimethyllinker or disulfide-containing linkers. Non-cleavable linkers tend toremain covalently associated with at least one amino acid of theantibody and the drug upon internalization by and degradation within thetarget cell. An exemplary non-cleavable linker is MCC.

Drugs

In certain embodiments, the antibody is conjugated to a chemotherapeuticagent. Examples of chemotherapeutic agents include alkylating agents,such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines, such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics, such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin .gamma1 and calicheamicin thetaI, see, e.g., Angew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites, such as methotrexate and5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens, such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals, such asaminoglutethimide, mitotane, trilostane; folic acid replenisher, such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids, such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;siRNA and pharmaceutically acceptable salts, acids or derivatives of anyof the above. Other chemotherapeutic agents that can be used with thepresent invention are disclosed in US Publication No. 20080171040 or USPublication No. 20080305044 and are incorporated in their entirety byreference.

It is contemplated that an antibody may be conjugated to two or moredifferent chemotherapeutic agents or a pharmaceutical composition maycomprise a mixture of antibodies wherein the antibody component isidentical except for being conjugated to a different chemotherapeuticagent. Such embodiments may be useful for targeting multiple biologicalpathways with a target cell.

In preferred embodiments, the ADC comprises an antibody conjugated toone or more maytansinoid molecules, which are mitotic inhibitors thatact by inhibiting tubulin polymerization. Maytansinoids, includingvarious modifications, are described in U.S. Pat. Nos. 3,896,111;4,151,042; 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; 4,371,533; and WO 2009/099728. Maytansinoid drugmoieties may be isolated from natural sources, produced usingrecombinant technology, or prepared synthetically. Exemplarymaytansinoids include C-19-dechloro (U.S. Pat. No. 4,256,746),C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,307,016 and 4,361,650), C-20-demethoxy (or C-20-acyloxy (—OCOR),+/−dechrolo (U.S. Pat. No. 4,294,757), C-9-SH (U.S. Pat. No. 4,424,219),C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598),C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254), C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866), C-15-methoxy(U.S. Pat. Nos. 4,313,946 and 4,315,929), C-18-N-demethyl (U.S. Pat.Nos. 4,362,663 and 4,322,348), and 4,5-deoxy (U.S. Pat. No. 4,371,533).

Various positions on maytansinoid compounds may be used as the linkageposition, depending upon the type of link desired. For example, forforming an ester linkage, the C-3 position having a hydroxyl group, theC-14 position modified with hydroxymethyl, the C-15 position modifiedwith a hydroxyl a group, and the C-20 position having a hydroxyl groupare all suitable (U.S. Pat. Nos. 5,208,020, RE39,151, and 6,913,748; USPatent Appl. Pub. Nos. 20060167245 and 20070037972, and WO 2009099728).

Preferred maytansinoids include those known in the art as DM1, DM3, andDM4 (US Pat. Appl. Pub. Nos. 2009030924 and 20050276812, incorporatedherein by reference).

ADCs containing maytansinoids, methods of making such ADCs, and theirtherapeutic use are disclosed in U.S. Pat. Nos. 5,208,020 and 5,416,064,US Pat. Appl. Pub. No. 20050276812, and WO 2009099728 (all incorporatedby reference herein). Linkers that are useful for making maytansinoidADCs are know in the art (U.S. Pat. No. 5,208,020 and US Pat. Appl. Pub.Nos. 2005016993 and 20090274713; all incorporated herein by reference).Maytansinoid ADCs comprising an SMCC linker may be prepared as disclosedin US Pat. Publ. No. 2005/0276812.

Effector Function-Enhanced Antibodies

One of the functions of the Fc portion of an antibody is to communicateto the immune system when the antibody binds its target. This isconsidered “effector function.” Communication leads toantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and/or complement dependent cytotoxicity(CDC). ADCC and ADCP are mediated through the binding of the Fc to Fcreceptors on the surface of cells of the immune system. CDC is mediatedthrough the binding of the Fc with proteins of the complement system,e.g., C1q.

The IgG subclasses vary in their ability to mediate effector functions.For example, IgG1 is much superior to IgG2 and IgG4 at mediating ADCCand CDC. Thus, in embodiments wherein a cell expressing ST2 is targetedfor destruction, an anti-ST2 IgG1 antibody would be preferred.

The effector function of an antibody can be increased, or decreased, byintroducing one or more mutations into the Fc. Embodiments of theinvention include antigen binding proteins, e.g., antibodies, having anFc engineered to increase effector function (U.S. Pat. No. 7,317,091 andStrohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated hereinby reference in its entirety). Exemplary IgG1 Fc molecules havingincreased effector function include (based on the Kabat numberingscheme) those have the following substitutions: S239D/I332E

S239D/A330S/I332E

S239D/A330L/I332E

S298A/D333A/K334A

P247I/A339D

P247/A339Q

D280H/K290S

D280H/K290S/S298D

D280H/K290S/S298V

F243L/R292P/Y300L

F243L/R292P/Y300L/P396L

F243L/R292P/Y300L/V305I/P396L

G236A/S239D/I332E

K326A/E333A

K326W/E333S

K290E/S298G/T299A

K290N/S298G/T299A

K290E/S298G/T299A/K326E

K290N/S298G/T299A/K326E

Further embodiments of the invention include antigen binding proteins,e.g., antibodies, having an Fc engineered to decrease effector function.Exemplary Fc molecules having decreased effector function include (basedon the Kabat numbering scheme) those have the following substitutions:

N297A (IgG1)

L234A/L235A (IgG1)

V234A/G237A (IgG2)

L235A/G237A/E318A (IgG4)

H268Q/V309L/A330S/A331S (IgG2)

C220S/C226S/C229S/P238S (IgG1)

C226S/C229S/E233P/L234V/L235A (IgG1)

L234F/L235E/P331S (IgG1)

S267E/L328F (IgG1)

Another method of increasing effector function of IgG Fc-containingproteins is by reducing the fucosylation of the Fc. Removal of the corefucose from the biantennary complex-type oligosaccharides attached tothe Fc greatly increased ADCC effector function without altering antigenbinding or CDC effector function. Several ways are known for reducing orabolishing fucosylation of Fc-containing molecules, e.g., antibodies.These include recombinant expression in certain mammalian cell linesincluding a FUT8 knockout cell line, variant CHO line Lec13, rathybridoma cell line YB2/0, a cell line comprising a small interferingRNA specifically against the FUT8 gene, and a cell line coexpressingβ-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II.Alternatively, the Fc-containing molecule may be expressed in anon-mammalian cell such as a plant cell, yeast, or prokaryotic cell,e.g., E. coli. Thus, in certain embodiments of the invention, acomposition comprises an antibody, e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,Ab7, Ab8, Ab9, Ab10, or Ab11 having reduced fucosylation or lackingfucosylation altogether.

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of one or a plurality ofthe antigen binding proteins of the invention together with apharmaceutically effective diluents, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In certain embodiments, the antigenbinding protein is an antibody. Pharmaceutical compositions of theinvention include, but are not limited to, liquid, frozen, andlyophilized compositions.

Preferably, formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of a ST2 antigen binding protein, e.g, a ST2-binding antibody,are provided.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine, proline, or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins of the invention. In certain embodiments, theprimary vehicle or carrier in a pharmaceutical composition may be eitheraqueous or non-aqueous in nature. For example, a suitable vehicle orcarrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. In specific embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,and may further include sorbitol or a suitable substitute therefor. Incertain embodiments of the invention, ST2 antigen binding proteincompositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the ST2 antigen binding protein product may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art. The formulation components are present preferablyin concentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired ST2 antigen binding protein in a pharmaceutically acceptablevehicle. A particularly suitable vehicle for parenteral injection issterile distilled water in which the ST2 antigen binding protein isformulated as a sterile, isotonic solution, properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads or liposomes, that may provide controlled orsustained release of the product which can be delivered via depotinjection. In certain embodiments, hyaluronic acid may also be used,having the effect of promoting sustained duration in the circulation. Incertain embodiments, implantable drug delivery devices may be used tointroduce the desired antigen binding protein.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, ST2 antigen binding proteins areadvantageously formulated as a dry, inhalable powder. In specificembodiments, ST2 antigen binding protein inhalation solutions may alsobe formulated with a propellant for aerosol delivery. In certainembodiments, solutions may be nebulized. Pulmonary administration andformulation methods therefore are further described in InternationalPatent Application No. PCT/US94/001875, which is incorporated byreference and describes pulmonary delivery of chemically modifiedproteins.

It is also contemplated that formulations can be administered orally.ST2 antigen binding proteins that are administered in this fashion canbe formulated with or without carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofthe ST2 antigen binding protein. Diluents, flavorings, low melting pointwaxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders may also be employed.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving ST2 antigen bindingproteins in sustained- or controlled-delivery formulations. Techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.See, for example, International Patent Application No. PCT/US93/00829,which is incorporated by reference and describes controlled release ofporous polymeric microparticles for delivery of pharmaceuticalcompositions. Sustained-release preparations may include semipermeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Sustained release matrices may include polyesters,hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 andEuropean Patent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Aspects of the invention includes self-buffering ST2 antigen bindingprotein formulations, which can be used as pharmaceutical compositions,as described in international patent application WO 06138181A2(PCT/US2006/022599), which is incorporated by reference in its entiretyherein.

As discussed above, certain embodiments provide ST2 antigen bindingproteins protein compositions, particularly pharmaceutical ST2 antigenbinding protein compositions, that comprise, in addition to the ST2antigen binding protein, one or more excipients such as thoseillustratively described in this section and elsewhere herein.Excipients can be used in the invention in this regard for a widevariety of purposes, such as adjusting physical, chemical, or biologicalproperties of formulations, such as adjustment of viscosity, and orprocesses of the invention to improve effectiveness and or to stabilizesuch formulations and processes against degradation and spoilage due to,for instance, stresses that occur during manufacturing, shipping,storage, pre-use preparation, administration, and thereafter.

A variety of expositions are available on protein stabilization andformulation materials and methods useful in this regard, such as Arakawaet al., “Solvent interactions in pharmaceutical formulations,” PharmRes. 8(3): 285-91 (1991); Kendrick et al., “Physical stabilization ofproteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEINFORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al.,“Surfactant-protein interactions,” Pharm Biotechnol. 13: 159-75 (2002),each of which is herein incorporated by reference in its entirety,particularly in parts pertinent to excipients and processes of the samefor self-buffering protein formulations in accordance with the currentinvention, especially as to protein pharmaceutical products andprocesses for veterinary and/or human medical uses.

Salts may be used in accordance with certain embodiments of theinvention to, for example, adjust the ionic strength and/or theisotonicity of a formulation and/or to improve the solubility and/orphysical stability of a protein or other ingredient of a composition inaccordance with the invention.

As is well known, ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface and by shieldingcharged and polar groups in the protein and reducing the strength oftheir electrostatic interactions, attractive, and repulsiveinteractions. Ions also can stabilize the denatured state of a proteinby binding to, in particular, the denatured peptide linkages (—CONH) ofthe protein. Furthermore, ionic interaction with charged and polargroups in a protein also can reduce intermolecular electrostaticinteractions and, thereby, prevent or reduce protein aggregation andinsolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating pharmaceuticalcompositions in accordance with the invention. One example is theHofmeister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic.” Destabilizingsolutes are referred to as “chaotropic.” Kosmotropes commonly are usedat high concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofmeister series.

Free amino acids can be used in ST2 antigen binding protein formulationsin accordance with various embodiments of the invention as bulkingagents, stabilizers, and antioxidants, as well as other standard uses.Lysine, proline, serine, and alanine can be used for stabilizingproteins in a formulation. Glycine is useful in lyophilization to ensurecorrect cake structure and properties. Arginine may be useful to inhibitprotein aggregation, in both liquid and lyophilized formulations.Methionine is useful as an antioxidant.

Polyols include sugars, e.g., mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.

Among polyols useful in select embodiments of the invention is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations. It ensures structural stability to the cake. It isgenerally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucroseare among preferred agents for adjusting tonicity and as stabilizers toprotect against freeze-thaw stresses during transport or the preparationof bulks during the manufacturing process. Reducing sugars (whichcontain free aldehyde or ketone groups), such as glucose and lactose,can glycate surface lysine and arginine residues. Therefore, theygenerally are not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred polyols of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard.

Embodiments of the ST2 antigen binding protein formulations furthercomprise surfactants. Protein molecules may be susceptible to adsorptionon surfaces and to denaturation and consequent aggregation atair-liquid, solid-liquid, and liquid-liquid interfaces. These effectsgenerally scale inversely with protein concentration. These deleteriousinteractions generally scale inversely with protein concentration andtypically are exacerbated by physical agitation, such as that generatedduring the shipping and handling of a product.

Surfactants routinely are used to prevent, minimize, or reduce surfaceadsorption. Useful surfactants in the invention in this regard includepolysorbate 20, polysorbate 80, other fatty acid esters of sorbitanpolyethoxylates, and poloxamer 188.

Surfactants also are commonly used to control protein conformationalstability. The use of surfactants in this regard is protein-specificsince, any given surfactant typically will stabilize some proteins anddestabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine. Consequently,polysorbates should be used carefully, and when used, should be employedat their lowest effective concentration. In this regard, polysorbatesexemplify the general rule that excipients should be used in theirlowest effective concentrations.

Embodiments of ST2 antigen binding protein formulations further compriseone or more antioxidants. To some extent deleterious oxidation ofproteins can be prevented in pharmaceutical formulations by maintainingproper levels of ambient oxygen and temperature and by avoiding exposureto light. Antioxidant excipients can be used as well to preventoxidative degradation of proteins. Among useful antioxidants in thisregard are reducing agents, oxygen/free-radical scavengers, andchelating agents. Antioxidants for use in therapeutic proteinformulations in accordance with the invention preferably arewater-soluble and maintain their activity throughout the shelf life of aproduct. EDTA is a preferred antioxidant in accordance with theinvention in this regard.

Antioxidants can damage proteins. For instance, reducing agents, such asglutathione in particular, can disrupt intramolecular disulfidelinkages. Thus, antioxidants for use in the invention are selected to,among other things, eliminate or sufficiently reduce the possibility ofthemselves damaging proteins in the formulation.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease. Mg⁺², Mn⁺², and Zn⁺², however,can destabilize rhDNase. Similarly, Ca⁺² and Sr⁺² can stabilize FactorVIII, it can be destabilized by Mg⁺², Mn⁺² and Zn⁺², Cu⁺² and Fe⁺², andits aggregation can be increased by Al⁺³ ions.

Embodiments of the ST2 antigen binding protein formulations furthercomprise one or more preservatives. Preservatives are necessary whendeveloping multi-dose parenteral formulations that involve more than oneextraction from the same container. Their primary function is to inhibitmicrobial growth and ensure product sterility throughout the shelf-lifeor term of use of the drug product. Commonly used preservatives includebenzyl alcohol, phenol and m-cresol. Although preservatives have a longhistory of use with small-molecule parenterals, the development ofprotein formulations that includes preservatives can be challenging.Preservatives almost always have a destabilizing effect (aggregation) onproteins, and this has become a major factor in limiting their use inmulti-dose protein formulations. To date, most protein drugs have beenformulated for single-use only. However, when multi-dose formulationsare possible, they have the added advantage of enabling patientconvenience, and increased marketability. A good example is that ofhuman growth hormone (hGH) where the development of preservedformulations has led to commercialization of more convenient, multi-useinjection pen presentations. At least four such pen devices containingpreserved formulations of hGH are currently available on the market.NORDITROPIN (somatropin) (liquid, Novo Nordisk), NUTROPIN AQ(somatropin) (liquid, Genentech) & GENOTROPIN (somatropin)(lyophilized—dual chamber cartridge, Pharmacia & Upjohn) contain phenolwhile SOMATROPE (somatropin) (Eli Lilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation anddevelopment of preserved dosage forms. The effective preservativeconcentration in the drug product must be optimized. This requirestesting a given preservative in the dosage form with concentrationranges that confer anti-microbial effectiveness without compromisingprotein stability.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability should bemaintained over the entire product shelf-life (.about. 18 to 24 months).An important point to note is that preservative effectiveness should bedemonstrated in the final formulation containing the active drug and allexcipient components.

ST2 antigen binding protein formulations generally will be designed forspecific routes and methods of administration, for specificadministration dosages and frequencies of administration, for specifictreatments of specific diseases, with ranges of bio-availability andpersistence, among other things. Formulations thus may be designed inaccordance with the invention for delivery by any suitable route,including but not limited to orally, aurally, opthalmically, rectally,and vaginally, and by parenteral routes, including intravenous andintraarterial injection, intramuscular injection, and subcutaneousinjection.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of an ST2 antigen bindingprotein-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication for which the ST2 antigen binding protein is being used,the route of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient. In certain embodiments, the clinician may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about30 mg/kg or more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 1.0 μg/kg up to about 20 mg/kg,optionally from 10 μg/kg up to about 10 mg/kg or from 100 μg/kg up toabout 5 mg/kg.

A therapeutic effective amount of a ST2 antigen binding proteinpreferably results in a decrease in severity of disease symptoms, in anincrease in frequency or duration of disease symptom-free periods, or ina prevention of impairment or disability due to the disease affliction.

Pharmaceutical compositions may be administered using a medical device.Examples of medical devices for administering pharmaceuticalcompositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196;4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824;4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163,all incorporated by reference herein.

Methods of Diagnosing or Treating a ST2-Associated Disease or Disorder

The ST2 antigen binding proteins of the invention are particularlyuseful for detecting ST2 in a biological sample. In certain embodiments,a biological sample obtained from a patient is contacted with a ST2antigen binding protein. Binding of the ST2 antigen binding protein toST2 is then detected to determine the presence or relative amount of ST2in the sample. Such methods may be useful in diagnosing or determiningpatients that are amenable to treatment with a ST2 antigen bindingprotein.

In certain embodiments, a ST2 antigen binding protein of the inventionis used to diagnose, detect, or treat an autoimmune or inflammatorydisorder. In treating autoimmune or inflammatory disorders, the ST2antigen binding protein may target ST2-expressing cells of the immunesystem for destruction and/or may block the interaction of ST2 withIL-33.

Disorders that are associated with IL-33-mediated signaling areparticularly amenable to treatment with one or more ST2 antigen bindingproteins disclosed herein. Such disorders include, but are not limitedto, inflammation, autoimmune disease, paraneoplastic autoimmunediseases, cartilage inflammation, fibrotic disease and/or bonedegradation, arthritis, rheumatoid arthritis, juvenile arthritis,juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoidarthritis, polyarticular juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenileenteropathic arthritis, juvenile reactive arthritis, juvenile Reter'sSyndrome, SEA Syndrome (Seronegativity, Enthesopathy, ArthropathySyndrome), juvenile dermatomyositis, juvenile psoriatic arthritis,juvenile scleroderma, juvenile systemic lupus erythematosus, juvenilevasculitis, pauciarticular rheumatoid arthritis, polyarticularrheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosingspondylitis, enteropathic arthritis, reactive arthritis, Reter'sSyndrome, SEA Syndrome (Seronegativity, Enthesopathy, ArthropathySyndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemiclupus erythematosus, vasculitis, myolitis, polymyolitis,dermatomyolitis, polyarteritis nodossa, Wegener's granulomatosis,arteritis, ploymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis,primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome,psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis,pustular psoriasis, erythrodermic psoriasis, dermatitis, atopicdermatitis, atherosclerosis, lupus, Still's disease, Systemic LupusErythematosus (SLE), myasthenia gravis, inflammatory bowel disease(IBD), Crohn's disease, ulcerative colitis, celiac disease, multipleschlerosis (MS), asthma, COPD, rhinosinusitis, rhinosinusitis withpolyps, eosinophilic esophogitis, eosinophilic bronchitis,Guillain-Barre disease, Type I diabetes mellitus, thyroiditis (e.g.,Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmunehepatitis, GVHD, transplantation rejection, kidney damage, and the like.

In preferred embodiments, the autoimmune or inflammatory disorder isasthma, atopic dermatitis, chronic obstructive pulmonary disease,pulmonary fibrosis, sepsis and trauma, HIV infection, systemic lupuserythematosus, inflammatory bowel disease, rheumatoid arthritis,sclerosis, Wegener's granulomatosis, Behchet disease, cardiovasculardisease, rhinosinusitis, nasal polyposis, and eosinophilic bronchitis.

In certain embodiments, a ST2 antigen binding protein of the inventionis used to diagnose, detect, or treat a cancer or tumorigenic disorder.In treating a cancer or tumorigenic disorder, the ST2 antigen bindingprotein may target ST2-expressing cells for destruction and/or may blockthe interaction of ST2 with IL-33, thereby reducing IL-33 mediatedsignaling. For example, high soluble ST2 expression is associated withimproved survival in breast cancer patients. (Prechtel et al, Lab Invest(2001) 81:159-165) Because soluble ST2 binds and blocks IL-33-mediatedsignaling, it is contemplate that the ST2 antigen binding proteins thatblock IL-33-mediated signaling would be useful in promoting improvedsurvival in breast cancer patients. Cancer or tumorigenic disorders thatmay be diagnosed, detected or treated with an ST2 antigen bindingprotein include, but are not limited to, solid tumors generally, lungcancer, ovarian cancer, breast cancer, prostate cancer, endometrialcancer, renal cancer, esophageal cancer, pancreatic cancer, squamouscell carcinoma, uveal melanoma, cervical cancer, colorectal cancer,bladder, brain, pancreatic, head, neck, liver, leukemia, lymphoma andHodgkin's disease, multiple myeloma, melanoma, gastric cancer,astrocytic cancer, stomach, and pulmonary adenocarcinoma.

The antigen binding proteins may be used to inhibit tumor growth,progression, and/or metastasis. Such inhibition can be monitored usingvarious methods. For instance, inhibition can result in reduced tumorsize and/or a decrease in metabolic activity within a tumor. Both ofthese parameters can be measured by MRI or PET scans, for example.Inhibition can also be monitored by biopsy to ascertain the level ofnecrosis, tumor cell death, and the level of vascularity within thetumor. The extent of metastasis can be monitored using known methods.

EXAMPLES

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the presentinvention and are not intended to limit its scope.

Example 1 ST2 Antibodies are Efficacious in an Animal Model of Asthma

This example demonstrates that administering antibodies that bind ST2and inhibit IL-33-mediated signaling are efficacious in an animal modelof an inflammatory disease, i.e., asthma. A neutralizing mouse ST2 mAb(ST2 surrogate mAb) inhibited the activity of exogenously administeredIL-33 in vivo. Mice were administered 200 ng of recombinant mouse IL-33intranasally two hours after intravenous injection of 100 ug of anti-ST2mAb. The next day, bronchoalveolar lavage fluid (BALF) IL-5concentrations were measured by ELISA. Baseline IL-5 concentrations wereobtained from the BALF of mice treated with saline before salinechallenge. Maximum BALF IL-5 concentrations were obtained from isotypecontrol Ig-treated mice challenged with IL-33. Compared to isotypecontrol Ig treatment, ST2 mAb treatment significantly inhibitedIL-33-induced IL-5 in the BALF of both BALB/c and C57BL/6 mouse strains(FIG. 1).

The ST2 surrogate mAb was efficacious in a cockroach allergen(CRA)-induced model of asthma, with ST2 antibody-treated mice havingsignificantly fewer BALF eosinophils than isotype control Ig-treatedmice. BALB/c mice were challenged with 100 μg CRA on days 1, 3, 6, 8,10, and 13. Mice were injected with 250 μg of either anti-ST2 mAb orisotype control Ig on days 0, 7, and 13, with the day 13 antibodyinjection occurring before the final intranasal CRA challenge. On day14, the mice were anesthetized and subjected to lung lavage. BALF cellpopulations were enumerated and treatment with anti-ST2 mAb resulted inthe presence of significantly fewer total BALF cells, with eosinophilscomprising the significantly impacted cell population (FIG. 2).

Example 2 Production of Anti-ST2 Antibodies Using the Xenomouse®Platform

The generation of fully human antibodies directed against human ST2 wascarried out using XENOMOUSE® technology (U.S. Pat. Nos. 6,114,598;6,162,963; 6,833,268; 7,049,426; 7,064,244, which are incorporatedherein by reference in their entirety; Green et al., 1994, NatureGenetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Greenand Jakobovitis, 1998, J. Ex. Med. 188:483-495, Kellermann and Green,2002, Current Opinion in Biotechnology, 13:593-597

Immunizations of XMG2K, XMG4K and XMG4KL XENOMOUSE® animals were carriedout with either a polypeptide comprising the extracellular domain ofhuman ST2 fused to a human antibody Fc domain or with the human ST2-Fcfusion protein complexed with human IL-33. A suitable amount ofimmunogen (i.e., ten g/mouse of soluble ST2) was used for initialimmunization of XENOMOUSE® animals according to the methods disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. Following the initialimmunization, subsequent boost immunizations of immunogen (five μg/mouseof either soluble ST2 or ST2/IL33 complex) were administered on aschedule and for the duration necessary to induce a suitable titer ofanti-ST2 antibody in the mice. Titers were determined a suitable method,for example, ELISA or by fluorescence activated cell sorting (FACs).

Animals exhibiting suitable titers were identified, and lymphocytesobtained from draining lymph nodes and, when necessary, pooled for eachcohort. Lymphocytes were dissociated from lymphoid tissue by grinding ina suitable medium (for example, Dulbecco's Modified Eagle Medium; DMEM;obtainable from Invitrogen, Carlsbad, Calif.) to release the cells fromthe tissues, and suspended in DMEM. B cells were selected and/orexpanded using a suitable method, and fused with a suitable fusionpartner, for example, nonsecretory myeloma P3X63Ag8.653 cells (AmericanType Culture Collection CRL 1580; Kearney et al, J. Immunol. 123, 1979,1548-1550), using techniques that are known in the art.

In one fusion method, lymphocytes were mixed with fusion partner cellsat a ratio of 1:4. The cell mixture was gently pelleted bycentrifugation at 400×g for 4 minutes, the supernatant decanted, and thecell mixture gently mixed (for example, by using a 1 mL pipette). Fusionwas induced with PEG/DMSO (polyethylene glycol/dimethyl sulfoxide;obtainable from Sigma-Aldrich, St. Louis Mo.; 1 mL per million oflymphocytes). PEG/DMSO was slowly added with gentle agitation over oneminute followed, by one minute of mixing. IDMEM (DMEM without glutamine;2 mL per million of B cells), was then added over 2 minutes with gentleagitation, followed by additional IDMEM (8 mL per million B-cells) whichwas added over 3 minutes.

The fused cells were gently pelleted (400×g 6 minutes) and resuspendedin 20 mL Selection media (DMEM containing Azaserine and Hypoxanthine[HA] and other supplemental materials as necessary) per million B-cells.Cells were incubated for 20-30 minutes at 37° C. and then re-suspendedin 200 mL Selection media and cultured for three to four days in T175flasks prior to 96 well plating.

Cells were distributed into 96-well plates using standard techniques tomaximize clonality of the resulting colonies. After several days ofculture, supernatants were collected and subjected to screening assays.The hybridoma supernatants generated from mice immunized with theST2-Fc/IL33 complex were screened with an ELISA-based assay performedusing 96-well polystyrene ELISA plates passively coated overnight at 4°C. with 0.5 ug/mL of ST2-Flag/his complexed to human IL-33. To determineST-2 specific binding, a second ELISA screen was performed using 96-wellpolystyrene plates passively coated overnight at 4° C. with 10 ug/mL ofneutravidin. Plates were then washed and loaded with 0.5 ug/mLbiotinylated human IL33. This ELISA screen identified over 1200 anti-ST2specific binders.

Hybridoma supernatants generated from mice immunized with soluble ST2-Fcwere screened for ST2 antigen specific antibodies by FluorometricMicrovolume Assay Technology (FMAT) by screening against recombinantHEK293T cells transiently transfected with full-length human ST2 andcounter-screening against mock-transfected HEK293T cells. Briefly, thecells were seeded into 384-well FMAT plates in a volume of 40 ul/well ata density of 6,000 ST2 positive cells/well and 14,000 mock transfectedST2 negative cells/well. Hybridoma supernatant was then added andallowed to bind for 1 hour at room temperature followed by a wash andsecondary detection using anti-Human Fc-Cy5 secondary antibody. ThisFMAT screen identified over 2200 anti-ST2 specific binders fromhybridomas generated from mice immunized with the extracellular domainof ST2.

This combined panel of 3400 anti-ST2 specific hybridoma supernatantswere then further characterized for the ability to functional antagonizeST2 signalling using a Interferon-γ cytokine release assay. Briefly,either purified human peripheral blood mononuclear cells (PBMNCs) orpurified human NK cells were seeded into 96 well tissue culture platesand stimulated with human IL-33 and IL-12, inducing the release ofinterferon-gamma into the supernatant. Interferon-gamma levels in thesupernatant were quantified and were directly correlated to Il-33/ST2dependant signalling. Using this bioassay, hybridoma samples were testedfor the ability to block interferon-gamma release through blockade ofthe ST2 signalling pathway. This screen identified 578 hybridomasupernatants generated from the ST2-Fc immunization that inhibitedinterferon-gamma release by greater than 80%. In addition, 505 hybridomasupernatants generated from the ST2Fc/IL-33 complex immunization wereidentified that inhibited interferon-gamma release by greater than 70%.

This panel of 1083 hybridoma supernatants was then further characterizedfor cross-reactive binding to mouse and cynomolgus monkey ST2, forrelative affinity ranking by limited antigen ELISA, for biochemicalreceptor/ligand blocking by ELISA and for endogenous binding by FACsusing cell lines. The data generated in these secondary assays was usedto diversify the large panel into 2 sets of 40 hybridoma lines whichwere advanced to sub-cloning, scale-up and purification.

Example 3 K_(D) Determinations

In this example, the affinity of ST2-binding antibodies was determined.Surface plasmon resonance evaluations were carried out using a PROTEONXPR-36 optical biosensor equipped with a GLC sensor chip (Bio-Rad).Biosensor analysis was conducted at 25° C. in a HBS-EP+ (1×) buffersystem (10 mM HEPES pH 7.4, 150 mM NaCl, 3.0 mM EDTA, 0.05% SurfactantP20, GE Heathcare). All reagents were kept at 8° C. prior to injection.

Goat anti-human IgG (Fc fragment specific, Jackson ImmunoResearch) wasimmobilized to the sensor surface in the vertical direction via standardamine coupling to lanes 1-6 (˜4000 RU) and then blocked withethanolamine. The antibodies were captured (˜40-100 RU) in the verticaldirection to lanes 1-5. Vertical lane 6 was left blank and used forreference purposes. The data were collected in groups of 15 antibodies(three sets of 5).

The ST2 reagents (human or cyno) were prepared in running buffer to aconcentration of 25 nM and then diluted 3-fold to 309 pM. A singleinjection along the horizontal direction delivered a full concentrationseries of each ST2 molecule, using buffer to complete a row of sixsamples and provide an in-line blank for double-referencing the responsedata. The association (3 min) and dissociation (30 min) rates weremonitored at a flow rate of 100 uL/min.

The surface was regenerated at a flow rate of 100 uL/min with 10 mMglycine (pH 1.5, 30 uL).

The data were baseline corrected, cropped, aligned, reference subtracted(interspot), and then fit to a 1:1 binding model using PROTEON MANAGER(software) (version 2.1.2.05). The results are shown in Table 4.

TABLE 4 Antibody Analyte ka kd KD (pM) Antibody Analyte ka kd KD (pM)Ab12 cy ST2 2.50E+06 5.60E−05 22.5 Ab12 hu ST2 2.35E+06 3.41E−05 14.5Ab13 cy ST2 1.40E+06 1.80E−04 128.0 Ab13 hu ST2 1.30E+06 9.12E−05 70.3Ab14 cy ST2 3.57E+06 1.59E−03 445.0 Ab14 hu ST2 4.22E+06 2.57E−05 6.1Ab15 cy ST2 2.67E+06 6.23E−05 23.4 Ab15 hu ST2 1.83E+06 5.38E−05 29.3Ab16 cy ST2 2.61E+06 2.18E−04 83.7 Ab16 hu ST2 1.28E+06 1.47E−04 115.0Ab17 cy ST2 3.38E+06 1.43E−04 42.2 Ab17 hu ST2 2.86E+06 1.04E−04 36.4Ab18 cy ST2 3.16E+06 1.44E−04 45.7 Ab18 hu ST2 2.67E+06 1.19E−04 44.5Ab19 cy ST2 3.07E+06 1.59E−04 51.8 Ab19 hu ST2 2.81E+06 1.25E−04 44.5Ab20 cy ST2 2.61E+06 6.64E−05 25.5 Ab20 hu ST2 2.41E+06 5.68E−05 23.5Ab21 cy ST2 3.21E+06 4.92E−05 15.3 Ab21 hu ST2 2.83E+06 3.07E−05 10.8Ab22 cy ST2 2.87E+06 5.33E−05 18.6 Ab22 hu ST2 2.50E+06 4.05E−05 16.2Ab23 cy ST2 3.29E+06 3.23E−04 98.2 Ab23 hu ST2 2.70E+06 2.24E−04 83.1Ab24 cy ST2 2.03E+06 1.54E−04 75.9 Ab24 hu ST2 2.89E+06 1.50E−04 51.7Ab25 cy ST2 6.42E+06 5.75E−04 89.6 Ab25 hu ST2 4.00E+06 5.44E−04 136.0Ab26 cy ST2 5.65E+06 3.08E−04 54.5 Ab26 hu ST2 5.22E+06 2.97E−04 56.9Ab27 cy ST2 1.63E+06 3.75E−04 230.0 Ab27 hu ST2 1.35E+06 3.12E−04 230.0Ab28 cy ST2 2.97E+06 1.35E−05 4.5 Ab28 hu ST2 2.37E+06 1.98E−05 8.4 Ab29cy ST2 3.97E+05 9.45E−05 238.0 Ab29 hu ST2 3.76E+05 8.96E−05 238.0 Ab30cy ST2 3.09E+06 3.17E−05 10.2 Ab30 hu ST2 2.79E+06 2.71E−05 9.7 Ab31 cyST2 1.07E+06 2.08E−04 194.0 Ab31 hu ST2 8.78E+05 2.43E−04 277.0 Ab32 cyST2 4.81E+06 2.69E−04 55.8 Ab32 hu ST2 4.37E+06 2.63E−04 60.2 Ab33 cyST2 4.26E+06 3.31E−04 77.6 Ab33 hu ST2 4.04E+06 3.41E−04 84.4 Ab34 cyST2 2.78E+06 4.60E−05 16.5 Ab34 hu ST2 2.61E+06 3.19E−05 12.3 Ab35 cyST2 9.76E+05 1.00E−04 103.0 Ab35 hu ST2 8.17E+05 1.15E−04 141.0 Ab36 cyST2 4140000 0.000278 67.1 Ab36 hu ST2 4.12E+06 2.80E−04 68.1

The affinity of additional antibodies were determined using a slightlymodified Plasmon resonance protocol. The surface plasmon resonanceevaluations for the antibodies Ab1, Ab2, Ab3, and Ab4 were conducted at25° C. using a BIACORE 3000 instrument (Biacore International AB,Uppsala, Sweden) equipped with a CM5 sensor chip. Anti-Fcγ specificcapture antibodies were covalently immobilized to two flow cells on theCM4 chip using standard amine-coupling chemistry with HBS-EP ((10 mMHEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20, GEHeathcare) as the running buffer. Briefly, each flow cell was activatedwith a 1:1 (v/v) mixture of 0.1 M NHS and 0.4 M EDC. AffiniPure GoatAnti-Human IgG, Fcγ Fragment Specific antibody (Jackson ImmunoResearchInc. West Grove, Pa.) at 30 ug/ml in 10 mM sodium acetate, pH 5.0 wasimmobilized with a target level of 3,000 RUs on two flow cells. Residualreactive surfaces were deactivated with an injection of 1 Methanolamine. The running buffer was then switched to HBS-EP+0.1 mg/mlBSA for all remaining steps.

All antibodies were prepared in running buffer in triplicate and diluted3-fold, and injected so that a three minute injection at 10 μl/min overthe test flow cell resulted in approximately 75-90 response units ofantibody captured on the test flow cell surface. No antibody wascaptured on the control flow cell surface. Human or cyno ST2 at variousconcentrations (200 nM-0.0914 nM), along with buffer blanks were thenflown over the two flow cells. A flow rate of 50 ul/min was used and atwo minute association phase followed by a four min dissociation phase.After each cycle the surfaces were regenerated with a 50 uL injection of10 mM glycine pH 1.5. Fresh antibody was then captured on the test flowcell to prepare for the next cycle. A separate long dissociationexperiment (60 min) was performed in triplicate at a concentration of200 nM.

Data was double referenced by subtracting the control surface responsesto remove bulk refractive index changes, and then subtracting theaveraged buffer blank response to remove systematic artifacts from theexperimental flow cells. The ST2 data were processed and globally fit toa 1:1 interaction model with a local Rmax in BIA evaluation Software v4.1. (Biacore International AB, Uppsala, Sweden). Association (k_(a))and dissociation (k_(d)) rate constants were determined and used tocalculate the dissociation equilibrium constant (K_(D)). Thedissociation rate constants and dissociation equilibrium constants forAb1, Ab2, Ab3, and Ab4 are summarized in Table 5.

TABLE 5 Antibody ST2 k_(a) (1/Ms) k_(d) (1/s) K_(D) (pM) Ab1 Human1.43E+06 1.11E−04 77.7 Ab1 Cyno 1.69E+06 1.97E−04 117 Ab2 Human 3.33E+051.13E−05 33.9 Ab2 Cyno 3.60E+05 1.16E−05 32.2 Ab3 Human 4.00E+059.50E−05 238 Ab3 Cyno 6.74E+05 8.55E−05 127 Ab4 Human 2.35E+06 7.06E−04301 Ab4 Cyno 2.50E+06 1.29E−03 516

Example 4 pH-Sensitive Binding of Antibodies

Therapeutic antibodies that bind with decrease affinity at low pH totheir targets may have enhanced PK properties that will allow them to bedelivered less frequently or at lower doses. (Nat Biotechnol. 201028(11):1203-7 T. Igawa et al Antibody recycling by engineeredpH-dependent antigen binding improves the duration of antigenneutralization) This is due to target release by the antibody at the lowpH of the lyzosome, followed by subsequent target degradation andantibody recycling.

Biosensor analysis of the pH sensitive binding of the antibodies Ab1,Ab2, Ab3, and Ab4 was carried out on a Biacore 4000. The setup wassimilar to Example 3, where the K_(D) measurements of these antibodieswere conducted, except that data was fit for a duplicate injection of asingle concentration of 2.46 nM of α-ST2 antibody. The association (5min) rates at pH 7.4 and dissociation (10 min) rates at pH 5.5 and 7.4were monitored at a flow rate of 30 uL/min. The reference subtracteddata was fit to a 1:1 model in Scrubber. Several of the antibodiesdisplayed dramatically faster off rates at lower pH, as shown in Table6.

TABLE 6 pH 7.4/pH Estimated 5.5 kd fold Antibody pH k_(d) (1/s)* changeAb1 7.4 0.000134 88.1 Ab1 5.5 0.0118 Ab2 7.4 0.0000298 8.0 Ab2 5.50.000238 Ab3 7.4 0.0000273 2.9 Ab3 5.5 0.0000791 Ab4 7.4 0.000632 16.9Ab4 5.5 0.0107

Example 5 Antibody Cross-Competition

A common way to characterize epitopes is through competitionexperiments. Antibodies that compete with each other can be thought ofas binding the same site on the target. This example describes a methodof determining competition for binding to ST2 and the results of themethod when applied to a number of antibodies described herein.

Binning experiments can be conducted in a number of ways, and the methodemployed may have an effect on the assay results. Common to thesemethods is that ST2 is typically bound by one reference antibody andprobed by another. If the reference antibody prevents the binding of theprobe antibody then the antibodies are said to be in the same bin. Theorder in which the antibodies are employed is important. If antibody Ais employed as the reference antibody and blocks the binding of antibodyB the converse is not always true: antibody B used as the referenceantibody will not necessarily block antibody A. There are a number offactors in play here: the binding of an antibody can causeconformational changes in the target which prevent the binding of thesecond antibody, or epitopes which overlap but do not completely occludeeach other may allow for the second antibody to still have enoughhigh-affinity interactions with the target to allow binding. In general,if competition is observed in either order the antibodies are said tobin together, and if both antibodies can block each other then it islikely that the epitopes overlap more completely.

For this Example, a modification of the Multiplexed Binning methoddescribed by Jia, et al (J. Immunological Methods, 288 (2004) 91-98) wasused. Soluble ST2-FLAG His was used. Each Bead Code ofstreptavidin-coated LUMINEX beads (Luminex, #L100-L1XX-01, XX specifiesthe bead code) was incubated in 100 ul of 6 pg/bead biotinylatedmonovalent mouse-anti-human IgG capture antibody (BD Pharmingen,#555785) for 1 hour at room temperature in the dark, then washed 3× withPBSA, phosphate buffered saline (PBS) plus 1% bovine serum albumin(BSA). Each bead code was separately incubated with 100 ul of a 1:10dilution anti-ST2 antibody (Coating Antibody) for 1 hour then washed.The beads were pooled then dispensed to a 96-well filter plate(Millipore, #MSBVN1250). 100 ul of 2 ug/ml ST2 was added to half thewells and buffer to the other half and incubated for 1 hour then washed.100 ul of a 1:10 dilution anti-ST2 antibody (Detection Ab) was added toone well with ST2 and one well without ST2, incubated for 1 hour thenwashed. An irrelevant human-IgG (Jackson, #009-000-003) as well as a noantibody condition (blank) were run as negative controls. 20 ulPE-conjugated monovalent mouse-anti-human IgG (BD Pharmingen, #555787)was added to each well and incubated for 1 hour then washed. Beads wereresuspended in 75 ul PBSA and at least 100 events/bead code werecollected on the BioPlex instrument (BioRad).

Median Fluorescent Intensity (MFI) of the antibody pair without ST2 wassubtracted from signal of the corresponding reaction containing ST2. Forthe antibody pair to be considered bound simultaneously, and thereforein different bins, the value of the reaction had to meet twocriteria: 1) the values had to be 2 times greater than the coatingantibody paired with itself, the irrelevant or the blank, whichever washighest, and 2) the values had to be greater than the signal of thedetection antibody present with the irrelevant or the blank coated bead.A minimum of three bins were found as shown in Table 7 below.

TABLE 7 Bin Antibody Bin 1 Ab23 Ab17 Ab24 Ab25 Ab12 Ab36 Ab14 Ab18 Ab19Ab20 Ab33 Ab34 Ab1 Ab7 Ab3 Ab15 Ab16 Ab27 Ab5 Ab2 Ab8 Ab13 Ab30 Ab35Ab28 Bin 2 Ab9 Ab10 Ab11 Bin 3 Ab29

Example 6 IL-33-Blocking Assays

The mechanism of action of the ST2 antibodies was explored using twoAlphaScreens. In combination, the assays were used to determine if theantibodies could inhibit the association of IL-33 with ST2 or incontrast if the antibodies could specifically block the association ofthe co-receptors ST2 and AcP while still allowing IL-33 to associatewith ST2. AlphaScreen is an acronym for Amplified Luminescent ProximityHomogenous Assay screen.

In the first screen, antibodies were evaluated for the ability to blockan association between IL-33 and ST2. This assay measured the ability ofthe anti-ST2 antibodies to block the association of biotinylated humanIL-33 (coupled with a Streptavidin donor bead) with 6×histidine taggedhuman ST2 (coupled with a Ni-chelate acceptor bead). The IL-33/ST2AlphaScreen was conducted using 40 ul reactions in a 96 well half areaplate (Perkin Elmer). The assay buffer that was used for bothAlphaScreens contained 40 mM HEPES (pH=7.4), 1 mM CaCl2, 0.1% BSA, 0.05%Tween-20 and 100 mM NaCl. Each assay well contained 0.3 nM biotinylatedhuman IL-33, 0.3 nM human ST2-FH (FH stands for FLAG and 6× Histidinetags), 10 ug/ml Streptavidin coated donor beads (Perkin Elmer, Waltham,Mass.), 10 ug/ml Ni-chelate coated acceptor beads (Perkin Elmer) and12.5 ug/ml of an anti-ST2 Ab. After the addition of all assay componentsthe plates were incubated overnight in the dark at room temperature. Thenext day the plates were read on a 2103 Envision multilabel reader(Perkin Elmer). Laser excitation of the donor beads at 680 nm was usedto generate reactive oxygen that could initiate aluminescent/fluorescent cascade in the acceptor beads that were in closeproximity due to the interaction of the bead coupled proteins resultingin the emission of light which was detected at 570 nm.

In the second assay, antibodies were evaluated for the ability toinhibit the IL-33 mediated association of ST2 with the co-receptor AcP.This assay measured the ability of the anti-ST2 antibodies to block theIL-33 mediated association of biotinylated human ST2-Fc (coupled with aStreptavidin donor bead) with 6×histidine tagged human AcP (coupled witha Ni-chelate acceptor bead). The ST2/AcP AlphaScreen was conducted in 8ul reactions in a 384 well optiplate (Perkin Elmer). Each assay wellcontained 5 nM human IL-33, 5 nM biotinylated human ST2-Fc, 5 nM humanAcP-FH, 10 ug/ml Streptavidin coated donor beads, 10 ug/ml Ni-chelatecoated acceptor beads and 12.5 ug/ml of an anti-ST2 Ab. After theaddition of all assay components the plates were incubated overnight inthe dark at room temperature. The next day the plates were read on a2103 Envision multilable reader (Perkin Elmer) using the same parametersas above for the first assay.

The results of the two AlphaScreens are presented in Table 8 below. Theinhibition of each antibody is presented as the percentage of inhibitionof signal in the AlphaScreen using a given antibody at a concentrationof 12.5 ug/ml relative to the signal in the assay when no antibody wasincluded in the assay well. Some antibodies inhibited the ST2 and IL-33interaction more completely than they inhibited the ST2/IL-33/AcPinteraction and some antibodies inhibited the ST2/IL-33/AcP interactionmore completely than the ST2 and IL-33 interaction. All antibodiesinhibited the IL-33 interaction with ST2 by at least 50%.

Table 8 % inhib % inhib ST2-IL33 ST2-AcP AS AS Name 12.5 ug/ml 12.5ug/ml Ab6 98.5 71.2 Ab4 98.4 77.8 Ab9 75.9 93.1 Ab10 51.8 73.2 Ab1 98.186.9 Ab7 98.9 75.7 Ab3 98.8 68.7 Ab11 75.8 93.6 Ab5 96.3 33.8 Ab2 99.296.4

Example 7 In Vitro Human IL-33 Bioassay

Exemplary ST2 human mAbs were tested in a human bioassay utilizingpurified CD4+ T cells obtained from various donors stimulated with humanIL-33 and human IL-2. The procedure for the assay is as follows. Cellsare seeded at 250,000 cells per well in 60 ul volume in a 96 well roundbottom plate. After preincubation, add 30 ul of 4× mixture ofhuIL-2+huIL-33 to each well. Total volume in 96-well round bottom plateis 120 ul. Start antibodies at 20 ug/ml and do 1:3 dilutions to generate10 point curve. Make 4× in 30 ul. After preincubation of Abs with cells,add 30 ul of 4× mixture of huIL-2+huIL-33 to each well. 37° C., 5% CO2for 48 hours. Harvest supernatants. Analyze inhibition of IL-5 by huIL-5ELISA.

FIG. 3 shows ST2 mAbs in the inhibition of human IL-33-induced IL-5production from CD4+ T cells from various donors. The (-) line depictsthe positive control value of human IL-33 in combination with human IL-2without inhibition. The ( . . . . ) depicts the positive control valueof human IL-2. The (--) line depicts the media control value. Human CD4+T cells were preincubated for 30 minutes with anti-ST2 mAbs and thenstimulated for 48 hours with human IL-33 (4 ng/ml) and human IL-2 (10ng/ml). FIG. 3 shows that ST2 antibodies are able to inhibit humanIL-33-induced ST2 activation, as determined by IL-5 production from CD4+T cells. The ST2 antibodies were able to antagonize IL-33 induced IL-5production from CD4+ T cells with IC50s of approximately <100 nM. Table9 shows representative IC50 values.

TABLE 9 IC50 Ab (nM) 2 5.25 8 6.90 10 6.90 1 10.68 9 62.01 5 64.54 11479.86

Example 8 Cynomolgus Monkey CD4+ T-cell IFNγ Release Assay

Cynomolgus monkey peripheral blood mononuclear cells (PBMC) wereenriched from acid citrate dextrose (ACD) treated normal donorperipheral blood by ISOLYMPH (Gallard-Schlesinger Industries, Plainview,N.Y.) gradient centrifugation. Subsequent isolation of cynomolgus monkeyCD4+ T cells was performed using Miltenyi Biotec's cynomolgus monkeyCD4+ T cell Isolation Kit. Isolated cyno CD4+ T cells (2×10⁻⁵ cells/wellin 96 well plates) were incubated with purified monoclonal antibodies atvarious concentrations for 30 minutes at room temperature and thenstimulated with IL-33 (10 ng/mL), IL-2 (10 ng/mL), and IL-12p70 (50ng/mL) for eighty-four hours. The resulting cell-free cynomolgus monkeyCD4+ T cell culture supernatants were then analyzed for the presence ofcynomolgus monkey IFNγ by ELISA (example data is provided in Table 10).The potency of purified monoclonal antibodies was determined in thecynomolgus monkey CD4+ T cell IFNγ release assay from three separatedonors.

TABLE 10 IC-50 Values pM Ab1 15.82 Ab2 79.5 Ab3 15.15 Ab4 4.03 Ab5 12.9Ab6 47.1 Ab7 40.01 Ab8 158.07

Example 9 Human Eosinophil IL-8 Release Assay

Human erythrocytes and granulocytes were enriched from heparinized,normal donor peripheral blood by ISOLYMPH (Gallard-SchlesingerIndustries, Plainview, N.Y.) gradient centrifugation. The erythrocyteswere removed using ACK lysing buffer (Gibco, Carlsbad, Calif.).Subsequent isolation of eosinophils was performed using MiltenyiBiotec's Eosinophil Isolation Kit. Isolated eosinophils (2×10⁵cells/well in 96 well plates) were incubated with non-clonal or clonalsupernatants at several dilutions, or purified monoclonal antibodies atvarious concentrations for 30 minutes at room temperature and thenstimulated with IL-33 (2 ng/mL) and IL-3 (100 ng/mL) for three days. Theresulting cell-free eosinophil culture supernatants were then analyzedfor the presence of IL-8 by ELISA. Example data is shown in Table 11.The potency of purified monoclonal antibodies was determined in theeosinophil IL-8 release assay from three separate donors.

TABLE 11 IC-50's pM Ab1 51.45 Ab2 52.75 Ab3 50.38 Ab4 14.12 Ab5 73.27Ab6 63.02 Ab7 40.68 Ab8 3120

Example 10 Potency of Anti-ST2 Antibody Compared to CommerciallyAvailable Antibodies

Dose Response of Human IL-33 in Human NK Cell Assay

Primary CD56-positive human NK cells (5×10e4 cells) were treated withhuman IL-12 (1 ng/mL) plus increasing amounts of human IL-33, as shownin FIG. 4. Twenty-two hours later, cell-free supernatants were collectedand measured for IFN-γ concentration using a commercial assay (R&DSystems). 10 ng/mL IL-33 was used as the stimulation dose for the ST2antibody inhibition.

Antibody Inhibition of IL-33 Activity

Human NK cells were stimulated as above. Thirty minutes prior to IL-33and IL-12 addition, ST2 antibodies were added to cells at theconcentrations as indicated in FIG. 5. Twenty-two hours following IL-33treatment, cell-free supernatants were collected and measured for IFN-γconcentration using a commercial assay (R&D Systems). Clone names areindicated for the commercially available antibodies. Only Ab2 completelyinhibited the IL-33-dependent IFN-γ response and it was significantlymore potent than any of the commercially available huST2 antibodies. TheIC50 value corresponding to each antibody is shown in Table 12.

TABLE 12 Antibody IC50 (ug/ml) 2A5 ~608 HB12 7.700 B4E6 ~43.54 FB9~498.4 97203 0.3851 Ab2 0.04123

Example 11 Alanine/Arginine Scanning Mutagenesis of ST2

This Example characterizes ST2 antibodies based on the effect ofmutagenesis of ST2 on their ability to bind the target. Previous bindingdata indicated that ST2 domains 1 and 2 are primarily responsible forantibody binding for the panel of antibodies analyzed by ST2 scanningmutagenesis in this Example. As such, only domains 1 and 2 (D1D2) of ST2were considered structurally in the context of the full length ST2 inthe designs of mutation sites.

The ST2 and IL-33 complex model coordinates were obtained from Lingel etal., Structure (London, England: 1993). Elsevier Ltd 17, 1398-410. Theper residue sidechain solvent accessibility of ST2 was calculated in theMolecular Operating Environment (Molecular Operating Environment (MOE),2011.10; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite#910, Montreal, QC, Canada, H3A 2R7, 2011.). These solvent accessibilityvalues were then used in the selection of D1D2 surface residues formutagenesis by first selecting all D1D2 residues with sidechainexposures of at least 10 Å² or glycine residues with total exposures ofat least 10 Å². Glycine residues with a positive Phi angle were removed,as were proline residues, since mutations at such positions have ahigher probability of distorting the local protein structure. Cysteineresidues were also removed from the selection to maintain disulfidelinkages. Residue A82 was removed after visual inspection. This methodproduced a list of 140 residues for mutagenesis. All residues weremutated to arginine except for arginine and lysine residues, which weremutated to alanine.

All mutant constructs of parental His-Avi-tagged ST2 extracellulardomain (ECD), in pTT5 vector, were expressed in transiently transfected293-6E suspension cells (NRCC) in 24-well plates. In-vivo biotinylationwas achieved by co-transfection of BiR A in pTT5 vector. Thesupernatants were dialyzed with PBS to remove excess biotin.

BioPlex binding assay is used to measure binding of anti-ST2 antibodiesto point mutations of ST2. The biotinylated mutants were bound onto 80bead codes of streptavidin-coated beads (Luminex, #L100-L1XX-01, XXspecifies the bead code). The 80 bead codes allowed the multiplexing oftwo sets of 70 mutants for 140 total. Each set included 6 parentalcontrols, 3 irrelevant protein controls and 1 blank. Antibody binding tomutant protein was compared to antibody binding to the parental.

100 ul of a 1:7 dilution of the ST2 mutants, parental and controls or noprotein pre bound to the beads were washed 5× with PBS+1% BSA, pooledand aliquoted into a 96-well filter plate (Millipore) then washed again.100 ul anti-ST2 antibodies in 3-fold dilutions were added to triplicatewells, incubated for 1 hour at RT and washed. 100 ul of 1:500 dilutionof PE-conjugated anti-human IgG Fc (Jackson, #109-116-170) was added toeach well, incubated for 0.5 hour and washed. Beads were resuspended in75 uL, shaken for at least 3 mins, and read on the BioPlex.

Before running the binding assay, a validation experiment was conductedto assess the “bead region” to “bead region” (B-B) variability. In thevalidation experiment, all beads were conjugated with the same wild-typecontrol protein. Therefore, the difference between beads regions was dueto purely B-B variance and was not confounded by difference between wildtype and mutant proteins. The titration of antibody was run with twelvereplications in different wells.

The objective of this statistical analysis was to estimate the B-Bvariability of the estimated EC50 of binding curves. The estimated B-Bstandard deviation (SD) was then used to build the EC50 confidenceintervals of wild type and mutant proteins during curve comparisonexperiments.

A four-parameter logistic model was fitted to the binding data for eachbead region. The resulting “sumout.xls” file, containing curve qualitycontrol (QC) results and parameter estimates for top (max), bottom(min), Hillslope (slope), and natural log of EC50 (xmid) of the curves,was used as the raw data for the analysis. B-B variability for eachparameter was then estimated by fitting mixed effect model using SASPROC MIXED procedure. Only curves with “good” QC status were included inthe analysis. The final mixed effect model included only residual (i.e.individual bead regions) as random effect. Least squares means (LS-mean)for each parameter were estimated by the mixed effect model as well. B-BSD was calculated by taking square root of B-B variance. Fold changebetween LS-mean+2SD and LS-mean−2SD, which represent approximately upperand lower 97.5 percentile of the population, was also calculated.

To identify mutants that did not produce much response relative to WTcontrol, for each antibody, mutants where max (MFI) are less than 30% ofthe max(MFI) for WT control are identified and flagged as Hitmax.

The EC50s of the mutant binding curves and wild type binding curves werecompared. Statistically significant differences were identified as hitsfor further consideration. The curves with “nofit” or “badfit” flagswere excluded from this analysis.

Two sources of variations are considered in the comparison of EC50estimates, variation from the curve fit and the bead-bead variation.Wild types and mutants were linked to different beads, hence theirdifference are confounded with the bead-bead difference. The curve fitvariation is estimated by the standard error of the log EC50 estimates.Bead-bead variation is experimentally determined using an experimentwhere wild type controls were linked to each one of the beads. The beadvariation in EC50 estimates of wild type binding curve from thisexperiment is used to estimate the bead-bead variation in the actualmapping experiment.

The comparisons of two EC50s (in log scale) are conducted usingStudent's t-test. A t-statistics is calculated as the ratio betweendelta (the absolute differences between EC50 estimates) and the standarddeviation of delta. The variance of delta is estimated by the sum of thethree components, variance estimate of EC50 for mutant and wild typecurves in the nonlinear regression and two times the bead-bead varianceestimated from a separate experiment. The multiple of two for thebead-bead variance is due to the assumption that both mutant and wildtype beads have the same variance.

The degree of freedom of the standard deviation of delta was calculatedusing the Satterthwaite's (1946) approximation.

Individual p-values and confidence intervals (95% and 99%) were derivedbased on Student's t distribution for each comparison.

In the case of multiple wild type controls, a conservative approach wastaken by picking the wild type control that was most similar to themutant, i.e., picking the ones with the largest p-values.

Multiplicity adjustments are important to control the false positivewhile conducting a large number of tests simultaneously. Two forms ofmultiplicity adjustment were implemented for this analysis: family wiseerror (FWE) control and false discovery rate (FDR) control. The FWEapproach controls the probability that one or more hits are not real;FDR approach controls the expected proportion of false positive amongthe selected hits. The former approach is more conservative and lesspowerful than the latter one. There are many methods available for bothapproaches, for this analysis, we chose Hochberg's (1988) method for FWEanalysis and Benjamini-Hochberg's (1995) FDR method for FDR analysis.Adjusted p-values for both approaches are calculated either for eachantibody or the whole assay.

A mutant was selected as having an effect by the following criteriaif: 1) a bad fit or no fit results was returned for that mutant, 2) themutant was selected for the hitmax criteria, 3) the family wise errorpValue was less than 0.01, or 4) the Bmax value was greater than 200% ofparental. A hit was designated as an inhibitor if the effect reducedBmax or increased the EC50 value, a hit was designated as an activatorif it increased Bmax or decreased the EC50 value. 8 mutation wereexcluded from the hit list due to their effect on >90% of the antibodiestested, they are: K37A, R46A, D63R, V71R, G106R, K112A, N132R, Q137R,and Y141R.

The results of the analysis are provided in Tables 13 and 14.

TABLE 13 Ab Bin Inhibiting mutants Activating mutants Ab2 1 L14R, I15R,S33R, E43R, V47R, A62R, G65R, L53R, R72A, S73R T79R, D92R, D97R, V104R,G138R, N152R, V176R Ab3 1 S3R, E10R, I15R, S33R, E43R, V47R, A62R, D29R,L53R, V61R, R72A, G65R, F76R, T79R, D92R, D97R, V104R, T162R T124R,K131A, Q134R, G138R, F147R, V176R, V184R Ab32 1 S3R, I15R, Y32R, S33R,E43R, V47R, S50R, D29R, R72A K55A, A62R, G65R, T79R, D92R, V95R, D97R,V104R, E128R, Q134R, G138R, S146R F147R, V176R Ab33 1 S3R, I15R, Y32R,S33R, T35R, E43R, V47R, D29R, R72A S50R, K55A, A62R, G65R, T79R, D92R,V95R, D97R, V104R, E128R, Q134R, G138R, S146R, F147R, N152R, V176R Ab301 S3R, L14R, Y26R, S33R, T35R, E43R, V47R, R72A A62R, G65R, T79R, D92R,V104R,, G138R,, A143R, F47R, N152R, V176R, V184R Ab11 2 S50R, S175R W7R,E10R, L14R, Q21R, E43R, T79R, N110R, T177R, V184R, K185A Ab10 2 A49R,S50R, I70R S175R, S181R K4A, Q5R, W7R, E10R, L14R, I15R, Q21R,Y26R,E43R, T79R, M100R, K109A, N110R, T124R, K145A, T177R, V184R, K185A Ab293 N11R, V47R, S50R, Y67R, N83R, V104R, L120R, G138R, S139R, S146R,F147R, A172R

TABLE 14 Ab No Effect mutants Ab2 K1A, F2R, S3R, K4A, Q5R, S6R, W7R,L9R, E10R, N11R, E12R, A13R, V16R, R17A, R20A, Q21R, K23A, S25R, Y26R,V28R, D29R, Y31R, Y32R, Q34R, T35R, N36R, S38R, T41R, Q42R, R44A, N45R,A49R, S50R, G51R, Q52R, K55A, L57A, E60R, V61R, S64R, I66R, Y67R, T68R,I70R, T75R, F76R, N77R, R78A, G80R, Y81R, N83R, T85R, Y87R, K88A, K89A,Q90R, S91R, N94R, V95R, Y98R, M100R, Y101R, S102R, T103R, S107R, E108R,K109A, N110R, Y114R, T117R, D119R, L120R, Y121R, N122R, W123R, T124R,E128R, F130R, K131A, Q134R, A135R, S139R, R140A, R142A, A143R, H144R,K145A, S146R, F147R, V149, D151R, M154R, T155R, E156R, A158R, D160R,T162R, K164A, I166R, H167R, N168R, N170R, A172R, N173R, Y174R, S175R,T177R, R180A, S181R, T183R, V184R, K185A, D186R, E187R Ab3 K1A, F2R,K4A, Q5R, S6R, W7R, L9R, N11R, E12R, A13R, L14R, V16R, R17A, R20A, Q21R,K23A, S25R, Y26R, V28R, Y31R, Y32R, Q34R, T35R, N36R, S38R, T41R, Q42R,R44A, N45R, A49R, S50R, G51R, Q52R, K55A, L57A, E60R, S64R, I66R, Y67R,T68R, I70R, S73R, T75R, N77R, R78A, G80R, Y81R, N83R, T85R, Y87R, K88A,K89A, Q90R, S91R, N94R, V95R, Y98R, M100R, Y101R, S102R, T103R, S107R,E108R, K109A, N110R, Y114R, T117R, D119R, L120R, Y121R, N122R, W123R,E128R, F130R, A135R, S139R, R140A, R142A, A143R, H144R, K145A, S146R,V149, D151R, N152R, M154R, T155R, E156R, A158R, D160R, K164A,I166R,H167R, N168R, N170R, A172R, N173R, Y174R, S175R, T177R, R180A,S181R, T183R, K185A, D186R, E187R Ab32 K1A, F2R, K4A, Q5R, S6R, W7R,L9R, E10R, N11R, E12R, A13R, L14R, V16R, R17A, R20A, Q21R, K23A, S25R,Y26R, V28R, Y31R, Q34R, T35R, N36R, S38R, T41R, Q42R, R44A, N45R, A49R,G51R, Q52R, L53R, L57A, E60R, V61R, S64R, I66R, Y67R, T68R, I70R, S73R,T75R, F76R, N77R, R78A, G80R, Y81R, N83R, T85R, Y87R, K88A, K89A, Q90R,S91R, N94R, Y98R, M100R, Y101R, S102R, T103R, S107R, E108R, K109A,N110R, Y114R, T117R, D119R, L120R, Y121R, N122R, W123R, T124R, F130R,K131A, A135R, S139R, R140A, R142A, A143R, H144R, K145A, V149, D151R,N152R, M154R, T155R, E156R, A158R, D160R, T162R, K164A, I166R,H167R,N168R, N170R, A172R, N173R, Y174R, S175R, T177R, R180A, S181R, T183R,V184R, K185A, D186R, E187R Ab33 K1A, F2R, K4A, Q5R, S6R, W7R, L9R, E10R,N11R, E12R, A13R, L14R, V16R, R17A, R20A, Q21R, K23A, S25R, Y26R, V28R,Y31R, Q34R, N36R, S38R, T41R, Q42R, R44A, N45R, A49R, G51R, Q52R, L53R,L57A, E60R, V61R, S64R, I66R, Y67R, T68R, I70R, S73R, T75R, F76R, N77R,R78A, G80R, Y81R, N83R, T85R, Y87R, K88A, K89A, Q90R, S91R, N94R, Y98R,M100R, Y101R, S102R, T103R, S107R, E108R, K109A, N110R, Y114R, T117R,D119R, L120R, Y121R, N122R, W123R, T124R, F130R, K131A, A135R, S139R,R140A, R142A, A143R, H144R, K145A, V149, D151R, M154R, T155R, E156R,A158R, D160R, T162R, K164A, I166R,H167R, N168R, N170R, A172R, N173R,Y174R, S175R, T177R, R180A, S181R, T183R, V184R, K185A, D186R, E187RAb30 K1A, F2R, K4A, Q5R, S6R, W7R, L9R, E10R, N11R, E12R, A13R, I15R,V16R, R17A, R20A, Q21R, K23A, S25R, V28R, D29R, Y31R, Y32R, Q34R, N36R,S38R, T41R, Q42R, R44A, N45R, A49R, S50R, G51R, Q52R, L53R, K55R, L57A,E60R, V61R, S64R, I66R, Y67R, T68R, I70R, S73R, T75R, F76R, N77R, R78A,G80R, Y81R, N83R, T85R, Y87R, K88A, K89A, Q90R, S91R, N94R, V95R, D97R,Y98R, M100R, Y101R, S102R, T103R, S107R, E108R, K109A, N110R, Y114R,T117R, D119R, L120R, Y121R, N122R, W123R, T124R, E128R, F130R, K131A,Q134R, A135R, S139R, R140A, R142A, H144R, K145A, S146R, V149, D151R,M154R, T155R, E156R, A158R, D160R, T162R, K164A, I166R,H167R, N168R,N170R, A172R, N173R, Y174R, S175R, T177R, R180A, S181R, T183R, K185A,D186R, E187R Ab11 K1A, F2R, S3R, K4A, Q5R, S6R, L9R, N11R, E12R, A13R,I15R, V16R, R17A, R20A, K23A, S25R, Y26R, V28R, D29R, Y31R, Y32R, S33R,Q34R, T35R, N36R, S38R, T41R, Q42R, R44A, N45R, V47R, A49R, G51R, Q52R,L53R, K55R, L57A, E60R, V61R, A62R, S64R, G65R, I66R, Y67R, T68R, I70R,R72R, S73R, T75R, F76R, N77R, R78A, G80R, Y81R, N83R, T85R, Y87R, K88A,K89A, Q90R, S91R, D92R, N94R, V95R, D97R, Y98R, M100R, Y101R, S102R,T103R, V104R, S107R, E108R, K109A, Y114R, T117R, D119R, L120R, Y121R,N122R, W123R, T124R, E128R, F130R, K131A, Q134R, A135R, G138R, S139R,R140A, R142A, A143R, H144R, K145A, S146R, F147R, V149, D151R, N152R,M154R, T155R, E156R, A158R, D160R, T162R, K164A, I166R,H167R, N168R,N170R, A172R, N173R, Y174R, V176R, R180A, S181R, T183R, D186R, E187RAb10 K1A, F2R, S3R, S6R, L9R, N11R, E12R, A13R, V16R, R17A, R20A, K23A,S25R, V28R, D29R, Y31R, Y32R, S33R, Q34R, T35R, N36R, S38R, T41R, Q42R,R44A, N45R, V47R, G51R, Q52R, L53R, K55R, L57A, E60R, V61R, A62R, S64R,G65R, I66R, Y67R, T68R, I70R, R72R, S73R, T75R, F76R, N77R, R78A, G80R,Y81R, N83R, T85R, Y87R, K88A, K89A, Q90R, S91R, D92R, N94R, V95R, D97R,Y98R, Y101R, S102R, T103R, V104R, S107R, E108R, Y114R, T117R, D119R,L120R, Y121R, N122R, W123R, E128R, F130R, K131A, Q134R, A135R, G138R,S139R, R140A, R142A, A143R, H144R, S146R, F147R, V149, D151R, N152R,M154R, T155R, E156R, A158R, D160R, T162R, K164A, I166R, H167R, N168R,A172R, N173R, Y174R, V176R, R180A, T183R, D186R, E187R Ab29 K1A, F2R,S3R, K4A, Q5R, S6R, W7R, L9R, E10R, E12R, A13R, L14R, I15R, V16R, R17A,R20A, Q21R, K23A, S25R, Y26R, V28R, D29R, Y31R, Y32R, S33R, Q34R, T35R,N36R, S38R, T41R, Q42R, E43R, R44A, N45R, A49R, G51R, Q52R, L53R, K55R,L57A, E60R, V61R, A62R, S64R, G65R, I66R, T68R, I70R, R72R, S73R, T75R,F76R, N77R, R78A, T79R, G80R, Y81R, T85R, Y87R, K88A, K89A, Q90R, S91R,D92R, N94R, V95R, D97R, Y98R, M100R, Y101R, S102R, T103R, S107R, E108R,K109A, N110R, Y114R, T117R, D119R, Y121R, N122R, W123R, T124R, E128R,F130R, K131A, Q134R, A135R, R140A, R142A, A143R, H144R, K145A, V149,D151R, N152R, M154R, T155R, E156R, A158R, D160R, T162R, K164A,I166R,H167R, N168R, N170R, N173R, Y174R, S175R, V176R, T177R, R180A,S181R, T183R, V184R, K185R, D186R, E187R

Example 12 Hydrogen/Deuterium Exchange (HDX) Analysis

In this Example, Ab2 was bound to ST2 and the effect of binding on HDXdetermined.

Soluble ST2 protein (domains 1-3 containing amino acids 19-322 of SEQ IDNO:1) with both FLAG-tag and His-tag on the C-terminus was transientlyexpressed in mammalian 293-6E cells and purified with IMAC (immobilizedmetal ion affinity chromatography) and further purified by preparativeSEC (size exclusion chromatography). The protein was then concentratedto 3.5 mg/mL using ultra-filtration. ST2 antibody Ab2 was expressed inengineered CHO-CS9 cells and purified with protein A affinitychromatography followed by preparative SEC. Analytical SEC was used todetermine that a 0.75:1.00 molar ratio of antibody:antigen was optimalto ensure that the ST2 protein is fully bound to the antibody. Both thefree ST2 protein and the antigen-antibody complex were stored in PBSbuffer, pH 7.2.

The HDX experiment was performed on an automated HDX system (Zhang,Zhang et al. 2012). Briefly, the H/D exchange process starts withdiluting 5 uL of either the free ST2 protein solution (3.5 mg/mL) orST2-antibody complex (with jST2 concentration of 3.5 mg/mL,antigen:antibody ratio of 1:0.75) into 25 uL D₂O buffer in 100 mM PBS,pH 7.2, which was prepared by dissolving PBS tablet in D₂O water, at 25°C. The exchange reaction was allowed to insubate for various labelingduration (30 seconds, 2, 8, 30 min, and 2, 8 hours) for each HDXexperiment, and the labeling reaction was quenched by mixing 20 μLlabeling solution with 80 μL quenching/denaturation/reduction buffer(7.25 M urea and 625 mM tris(2-carboxyethyl)phosphine (TCEP), 0.45Mglycine, pH 2.7) at 1° C.

A 40 μL aliquot of quenched solution was transferred into 120 μL 0.4mg/mL porcine pepsin (Sigma, St. Louis, Mo.) solution. The digestionsolution was immediately injected into a sample loop at 1° C. and stayed6 min for full digestion. Furthermore, the digest was separated by C18columns (3 columns in series, BEH C18, 2.1 mm×5 mm, Waters Corp.,Milford, Mass.). The HPLC separation was performed at 1° C. with a 5-minACN gradient of 1-40%. The LC eluant was analyzed by an Orbitrap Elitemass spectrometer (Thermo Scientific, San Jose, Calif.) in adata-dependent LC-MS/MS experiment.

Deuterium labeling, quenching, proteolytic digestion and injection wereperformed on a LEAP HD-X PAL system controlled by LEAP Shells (LEAPTechnologies, Carrboro, N.C.).

The experiments were repeated three times and in each experiment, eachtime point was repeated twice. A standard peptide mixture was added tothe samples to track and correct back-exchange variability. The mixturecontains these three peptides: bradykinin, angiotensin I, and leucineenkephalin. A specially designed tetrapeptide PPPI (synthesized byAnaSpec, Fremont, Calif.) was used as second internal standard tominimize run to run variability. Digest of ST2 protein without H/Dexchange was also analyzed as control.

The resulting data files were processed with the MassAnalyzer program(Zhang, Zhang et al. 2012). The software identifies peptides from theantigen digest, calculates the exchange rate of each peptide, correctsfor back-exchange information obtained from the internal standards, andfits the data into a model that gives protection factors to eachresidue.

Comparison of the exchange profiles between free ST2 and antibody-boundST2 protein revealed two regions of interest and could be the potentialepitope. The two regions in the ST2 sequence were refined toIVRCPRQGKPSY (amino acids 33-44 of SEQ ID NO:1 corresponding to aminoacids 15-26 of mature ST2) and IVRSPTF (amino acids 88-94 of SEQ ID NO:1corresponding to amino acids 70-76 of mature ST2) by multiple overlappedpeptides. These regions were less protected when ST2 is in thefree-state, while the exchange rate decreases dramatically upon bindingof ST2 and the antibody. Furthermore, based on ST2 homology structuremodel shown in FIG. 6, these two sequence stretches occupy similarspatial locations on the exterior surface of the protein. These resultslead to the conclusion that the two peptides are involved in the bindingbetween Ab2 and ST2 protein.

Example 13 X-Ray Crystallography

In this Example, the crystal structure of the complex of an sc-dsFvfragment of Ab2 and ST2 provides the specific amino acid residues in theinteraction interface.

Ab2 sc-dsFv was expressed in BL21(DE3) Star cells. Briefly, the Ab2sc-dsFv contains the heavy chain variable domain connected to the lightchain variable domain by a linker (G₄S)₃. To further stabilize themolecule, a disulfide bond was engineered into the molecule by mutatingto cysteine position 44 of the heavy chain variable region and position101 of the light chain variable region. The protein was expressed as aninclusion body. The inclusion body was solubilized and refolded. Proteinwas further purified on size exclusion column (SEC) and ion-exchangeMonoQ column, and then polished on SEC.

ST2 was expressed in 293S cells. The protein was purified by Ni-affinitycolumn and deglycosylated with EndoH. Protein was further purified onSEC.

The complex of Ab2 sc-dsFv and ST2 was formed by mixing Ab2 sc-dsFv andexcess ST2. The complex was purified on SEC and concentrated to 12 mg/mlfor crystallization. The protein complex was crystallized in 32-36%PEG400 and 0.1 M Hepes, pH 7.5-8.5, at 16° C. using sitting drop vapordiffusion method.

A 1.9 Å resolution dataset was collected at the Advanced Light Source,Lawrence Berkeley National Lab (Berkeley, Calif.). The data wasprocessed with MOSFLM (Leslie, 1992) and scaled by SCALA in CCP4 programsuite (Collaborative Computational Project, No 4. (1994)). The structurewas solved by molecular replacement method with program PHASER (McCoy etal. 2007) using the D1D2 domain of the published structure of IL-1RII(pdb code: 3O4O) and the variable domain of the Fab structure of Ab2 assearch models. Iterative structure refinement and model building werecarried out in with REFMAC5 (Murshudov et al., 1997) and COOT (Emsleyand Cowtan, 2004).

The interface analysis was carried out with program PISA, AREAMOL andNCONTACT in CCP4 package. The figures were prepared with Pymol (ThePyMOL Molecular Graphics System. Schrödinger).

The crystal structure of the ST2/Ab2 sc-dsFv complex was solved to aresolution of 1.9 Å. There are two independent pairs of ST2/Ab2 sc-dsFvcomplexes in the asymmetric unit (FIG. 7). Each complex consists of oneST2 molecule and one Ab2 sc-dsFv fragment. The ST2 molecule is made oftwo IgG-like domains (D1 and D2 domain). The Ab2 Fv domain utilizes allsix CDR loops of the heavy chain and light chain to interact with theST2 molecule (FIG. 8). For ST2, two loops (loop BC and loop FG) and theN-terminus of ST2 make direct interactions with the antibody. The totalburied solvent accessible surface area between ST2 and Ab2 sc-dsFv is1803 Å².

The interface between ST2 and Ab2 is highly charged. ST2 possess acluster of basic residues (Lys and Arg) in the D1 domain. Thispositive-charged surface patch complements the negative-charged patch onAb2 formed by clusters of acidic residues (Asp and Glu) in the CDRregions (FIG. 9A and FIG. 9B).

Two different methods were used to define the interface residues. In thefirst method, the interface residues were defined using solvent exposuredifference. The solvent accessible surface area of each residue in ST2(or Ab2 sc-dsFv) in the complex was calculated and compared to thesolvent accessible surface area of the corresponding residues in ST2 (orAb2 sc-dsFv) stripped from complex. All amino acids with differencegreater than 10% are listed in Table 15 and Table 16 for ST2 and Ab2,respectively. The surface exposure differences of Arg72 and Tyr81 of ST2are less than 10%. However, inspection of the complex structure revealedthat both residues form water-mediated hydrogen bonds with Ab2 heavychain residues and hence they are included in the list.

TABLE 15 Epitope residues as defined by solvent exposure differences:ST2. Residue numbers correspond to the position of the amino acid inmature ST2, i.e., amino acid 1 corresponds to amino acid 19 of SEQ IDNO: 1. Residue ASA in ASA in percentage Residue number complex apo ratiochange(%) LYS 1 68.5 196.9 0.348 65.2 PHE 2 48.4 106.2 0.456 54.4 PRO 197.9 10.8 0.731 26.9 ARG 20 0 79.6 0.000 100.0 GLN 21 81.7 98.1 0.83316.7 GLY 22 17.8 59.3 0.300 70.0 LYS 23 51.5 130.6 0.394 60.6 TYR 2632.4 56.6 0.572 42.8 ILE 70 16.6 30.9 0.537 46.3 VAL 71 1.7 5.3 0.32167.9 ARG 72 52.2 56.9 0.917 8.3 SER 73 18 22.2 0.811 18.9 PRO 74 70.9106.8 0.664 33.6 THR 75 3.8 84.6 0.045 95.5 PHE 76 0 134.3 0.000 100.0ASN 77 2.8 47.6 0.059 94.1 ARG 78 1.1 83.6 0.013 98.7 THR 79 7 37 0.18981.1 TYR 81 78.3 80.8 0.969 3.1 ASN77-NAG 502 148.2 249 0.595 40.5

TABLE 16 Paratope residues as defined by solvent HC residue ASA in ASAin percentage residue number complex apo ratio change TRP 33 1.9 32.20.059 94.1 ILE 50 0 8.3 0.000 100.0 ASP 57 52.8 59.6 0.886 11.4 ARG 5960.4 89.5 0.675 32.5 HIS 99 2 6.6 0.303 69.7 GLY 100 3.6 5.5 0.655 34.5THR 101 27.5 35.9 0.766 23.4 SER 102 48.8 97.6 0.500 50.0 SER 103 45.3111.8 0.405 59.5 ASP 104 23.1 93 0.248 75.2 TYR 105 1.9 75.4 0.025 97.5TYR 106 36.8 64.6 0.570 43.0 LC residue ASA in ASA in percentage residuenumber complex apo ratio change ASP 28 85.1 105.2 0.809 19.1 SER 30 11.950.9 0.234 76.6 ASN 31 12.5 47.1 0.265 73.5 TYR 32 1.1 109.9 0.010 99.0TYR 49 8.4 58.1 0.145 58.5 ASP 50 8.1 51.5 0.157 84.3 ASN 53 20.2 59.50.339 66.1 GLU 55 27.5 38.4 0.716 28.4 THR 56 116.9 127.4 0.918 8.2 ASP91 2 27.4 0.073 92.7 ASP 92 2.5 54.1 0.046 95.4 ASN 93 50.5 69.5 0.72727.3 PHE 94 54.9 110.9 0.495 50.5 LEU 96 2.9 7.8 0.372 62.8

In the second method, interface residues were selected that have atleast one atom within a predefined distance to its partner protein. Twoshells were defined based on different distance cutoff.

Core shell includes all residues with distance up to 5.0 Å.

Boundary shell includes all residues with distance longer than 5.0 Å butshorter than 8.0 Å.

The complete list of amino acid residues in each shell for ST2, theheavy and light chain of Ab2 are shown in Tables 17, 18 and 19,respectively. For residues that make hydrogen bond or salt bridges withits partner protein, the type of specific interactions are denoted inparenthesis after the residue (HB for hydrogen bond and SB for saltbridge).

TABLE 17 Epitope residues defined by distance cutoff: ST2. Residuenumbers correspond to the position of the amino acid in mature ST2,i.e., amino acid 1 corresponds to amino acid 19 of SEQ ID. NO: 1.Boundary Core (0-5 Å) (5-8 Å) l (LYS) (HR/SB) 3 (SER) 2 (PHE) 4 (LYS) 19(PRO) 18 (CYS) 20 (ARG) (HB/SB) 24 (PRO) 21 (GLN) 27 (THR) 22 (GLY) 28(VAL) 23 (LYS) (HB/SB) 31 (TYR) 26 (TYR) (HB) 68 (THR) 70 (ILE) 69 (CYS)71 (VAL) 80 (GLY) 72 (ARG) 81 (TYR) 73 (SER) 74 (PRO) 75 (THR) (HB) 76(PHE) 77 (ASN) (HB) 78 (ARG) (HB/SB) 79 (THR) (HB) 502 (NAG)-77(ASN)

TABLE 18 Paratope residues defined by distance cutoff: Ab2 heavy chain.core (0-5 Å) boundary (5-8 Å) 33 (TRP) 31 (ASN) 50 (ILE) 32 (TYR) 57(ASP) 34 (ILE) 59 (ARG) 47 (TRP) 99 (HIS) 52 (TYR) 100 (GLY) 55 (ASN)101 (THR) 58 (THR) 102 (SER) (HB) 98 (ARG) 103 (SER) (HB) 107 (GLY) 104(ASP) (HB/SB) 108 (LEU) 105 (TYR) (HB) 109 (ASP) 106 (TYR)

TABLE 19 Paratope residues defined by distance cutoff: Ab2 light chain.core (0-5 Å) boundary (5-8 Å) 28 (ASP) (HB) 2 (ILE) 29 (ILE) 27 (GLN) 30(SER) 33 (LEU) 31 (ASN) (HB) 34 (ASN) 32 (TYR) 46 (LEU) 49 (TYR) 52(SER) 50 (ASP) (HB/SB) 54 (LEU) 53 (ASN) 67 (SER) 55 (GLU) (HB/SB) 68(GLY) 56 (THR) 89 (GLN) 91 (ASP) (HB/SB) 90 (GLN) 92 (ASP) (HB/SB) 95(PRO) 93 (ASN) 96 (LEU) 94 (PHE) 96 (LEU)

Epitope residues that were defined by solvent exposure differences matchresidues of the core interaction groups defined by distance cut-off.Table 20 lists all hydrogen bond and salt bridge pairs within theinterface.

TABLE 20 Interaction pairs in the interface Ab2 Light Chain - ST2 ##Structure 1 Dist. Structure 2 Hydrogen Bonds 1 L:ASN 31 [ND2] 3.4 A:ARG20 [O] 2 L:GLU 55 [OE1] 2.7 A:LYS 1 [NZ] 3 L:ASP 50 [OD2] 2.8 A:ARG 20[NE] 4 L:ASP 50 [OD1] 3.0 A:ARG 20 [NH2] 5 L:ASP 28 [O] 2.9 A:LYS 23[NZ] 6 L:ASP 92 [OD1] 2.9 A:LYS 23 [NZ] 7 L:ASP 92 [OD1] 3.3 A:TYR 26[OH] 8 L:ASP 91 [O] 2.7 A:THR 75 [OG1] 9 L:ASP 91 [O] 3.0 A:ARG 78 [NH2]10 L:ASP 91 [OD2] 3.0 A:ARG 78 [NH2] Salt Bridges 1 L:GLU 55 [OE1] 2.7A:LYS 1 [NZ] 2 L:GLU 55 [OE2] 3.6 A:LYS 1 [NZ] 3 L:ASP 50 [OD1] 3.9A:ARG 20 [NE] 4 L:ASP 50 [OD2] 2.8 A:ARG 20 [NE] 5 L:ASP 50 [OD1] 3.0A:ARG 20 [NH2] 6 L:ASP 50 [OD2] 3.4 A:ARG 20 [NH2] 7 L:ASP 92 [OD2] 3.4A:LYS 23 [NZ] 8 L:ASP 92 [OD1] 2.9 A:LYS 23 [NZ] 9 L:ASP 91 [OD2] 3.0A:ARG 78 [NH2] Ab2 Heavy Chain - ST2 ## Structure 1 Dist. Structure 2Hydrogen Bonds 1 H:TYR 105 [N] 3.3 A:ASN 77 [O] 2 H:SER 102 [O] 2.6A:ASN 77 [ND2] 3 H:SER 103 [O] 2.7 A:THR 79 [OG1] 4 H:ASP 104 [OD1] 2.7A:LYS 1 [N] 5 H:ASP 104 [OD2] 2.8 A:THR 79 [N] 6 H:ASP 104 [OD2] 2.9A:ARG 20 [NH1] 7 H:TYR 105 [O] 2.8 A:ARG 20 [NH2] Salt Bridges 1 H:ASP104 [OD1] 2.7 A:LYS 1 [N] 2 H:ASP 104 [OD2] 3.2 A:LYS 1 [N] 3 H:ASP 104[OD2] 2.9 A:ARG 20 [NH1]

The ST2 epitope regions obtained from HDX-MS analysis of Example 12 areconfirmed by the crystallography data. The two epitopes (15-26 and70-76) from HDX were identified as epitopes in the higher resolutioncrystallography data. Specifically, Arg20, Gly22, Lys23 and Tyr26, aswell as Thr75 were identified to be close to the antibody with adistance of less than 3.4 Å. Additional residues that were identified tohave a distance to the antibody between 3.4 and 5 Å (Pro19, Gln21,Ile70, Val71, Arg72, Ser73 Pro74 and Phe76) were also covered in the HDXepitopes.

Overall, the results confirmed that ST2 epitope regions obtained fromboth HDX-MS and crystallography are consistent. The BC loop and FG loopin Domain 1 (see crystallography data) are the main interaction sites.

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What is claimed:
 1. An isolated ST2 antigen binding protein comprising:a) a light chain variable domain having at least 90% identity to theamino acid sequence set forth in SEQ ID NO:96; b) a heavy chain variabledomain having at least 90% identity to the amino acid sequence set forthin SEQ ID NO:30; or c) a light chain variable domain of a) and a heavychain variable domain of b).
 2. The ST2 antigen binding protein of claim1, wherein the light chain variable domain has at least 95% identity tothe amino acid sequence set forth in SEQ ID NO:96.
 3. The ST2 antigenbinding protein of claim 2, wherein the heavy chain variable domain hasat least 95% identity to the amino acid sequence set forth in SEQ IDNO:30.
 4. An isolated ST2 antigen binding protein, comprising: a) alight chain variable domain having no more than ten amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:96; b) a heavy chain variable domain having no morethan ten amino acid additions, deletions or substitutions from the aminoacid sequence set forth in SEQ ID NO:30; or c) the light chain variabledomain of a) and the heavy chain variable domain of b).
 5. The ST2antigen binding protein of claim 4, wherein the light chain variabledomain has no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:96. 6.The ST2 antigen binding protein of claim 5, wherein the heavy chainvariable domain has no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:30. 7.The ST2 antigen binding protein of any of claims 1-6, wherein the lightchain variable domain comprises the amino acid sequence set forth in SEQID NO:96.
 8. The ST2 antigen binding protein of claim 7, wherein theheavy chain variable domain comprises the amino acid sequence set forthin SEQ ID NO:30.
 9. An isolated ST2 antigen binding protein comprising:a light chain variable domain comprising: an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:107; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:118; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:129; or a heavy chain variable domaincomprising: an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:41; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:52; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:63.
 10. The ST2 antigen binding protein of claim 9, wherein the lightchain variable domain comprises an LCDR1 as set forth in SEQ ID NO:107;an LCDR2 sequence as set forth in SEQ ID NO:118; and an LCDR3 sequenceas set forth in SEQ ID NO:129; and the heavy chain variable domaincomprises an HCDR1 as set forth in SEQ ID NO:41; an HCDR2 sequence asset forth in SEQ ID NO:52; and an HCDR3 sequence as set forth in SEQ IDNO:63.
 11. The ST2 antigen binding protein of any of claim 1, 4, or 9,wherein the antigen binding protein is an antibody.
 12. The ST2 antigenbinding protein of claim 11, wherein the antibody is a human antibody.13. The ST2 antigen binding protein of claim 12 comprising a light chainand a heavy chain, wherein the light chain comprises the amino acidsequence set forth in SEQ ID NO:85 and the heavy chain comprises theamino acid sequence set forth in SEQ ID NO:19.
 14. An isolated ST2antibody comprising: a) a light chain variable domain having at least90% identity to the amino acid sequence set forth in SEQ ID NO:96; b) aheavy chain variable domain having at least 90% identity to the aminoacid sequence set forth in SEQ ID NO:30; or c) a light chain variabledomain of a) and a heavy chain variable domain of b).
 15. The isolatedST2 antibody of claim 14, wherein the light chain variable domain has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO:96.16. The isolated ST2 antibody of claim 15, wherein the heavy chainvariable domain has at least 95% identity to the amino acid sequence setforth in SEQ ID NO:30.
 17. An isolated ST2 antibody comprising: a) alight chain variable domain having no more than ten amino acidadditions, deletions or substitutions from the amino acid sequence setforth SEQ ID NO:96; b) a heavy chain variable domain having no more thanten amino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:30; or c) a light chain variable domainof a) and the heavy chain variable domain of b).
 18. The isolated ST2antibody of claim 17, wherein the light chain variable domain has nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth SEQ ID NO:96.
 19. The isolated ST2antibody of claim 18, wherein the heavy chain variable domain has nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth SEQ ID NO:30.
 20. An isolated ST2 antibodycomprising: a) a light chain variable domain comprising an LCDR1 havingno more than three amino acid additions, deletions, or substitutionsfrom the LCDR1 sequence set forth in SEQ ID NO:107; an LCDR2 having nomore than three amino acid additions, deletions, or substitutions fromthe LCDR2 sequence set forth in SEQ ID NO:118; and an LCDR3 having nomore than three amino acid additions, deletions, or substitutions fromthe LCDR3 sequence set forth in SEQ ID NO:129; b) a heavy chain variabledomain comprising an HCDR1 having no more than three amino acidadditions, deletions, or substitutions from the HCDR1 sequence set forthin SEQ ID NO:41; an HCDR2 having no more than three amino acidadditions, deletions, or substitutions from the HCDR2 sequence set forthin SEQ ID NO:52; and an HCDR3 having no more than three amino acidadditions, deletions, or substitutions from the HCDR3 sequence set forthin SEQ ID NO:63; or c) a light chain variable domain of a) and the heavychain variable domain of b).
 21. The isolated ST2 antibody of any ofclaim 14, 17, or 20, wherein said light chain variable region comprisesD28 or a conservative substitution thereof, I29 or a conservativesubstitution thereof, S30 or a conservative substitution thereof, N31 ora conservative substitution thereof, Y32 or a conservative substitutionthereof, Y49 or a conservative substitution thereof, D50 or aconservative substitution thereof, N53 or a conservative substitutionthereof, E55 or a conservative substitution thereof, T56 or aconservative substitution thereof, D91 or a conservative substitutionthereof, D92 or a conservative substitution thereof, N93 or aconservative substitution thereof, F94 or a conservative substitutionthereof, or L96 or a conservative substitution thereof.
 22. The isolatedST2 antibody of claim 21, wherein said light chain variable regioncomprises D28 or a conservative substitution thereof, N31 or aconservative substitution thereof, D50 or a conservative substitutionthereof, N53 or a conservative substitution thereof, E55 or aconservative substitution thereof, D91 or a conservative substitutionthereof, and D92 or a conservative substitution thereof.
 23. Theisolated ST2 antibody of claim 22, wherein said light chain variableregion comprises D28, N31, D50, N53, E55, D91, and D92.
 24. The isolatedST2 antibody of any of claim 14, 17, or 20, wherein said heavy chainvariable region comprises W33 or a conservative substitution thereof,I50 or a conservative substitution thereof, D57 or a conservativesubstitution thereof, R59 or a conservative substitution thereof, H99 ora conservative substitution thereof, G100 or a conservative substitutionthereof, T101 or a conservative substitution thereof, S102 or aconservative substitution thereof, S103 or a conservative substitutionthereof, D104 or a conservative substitution thereof, Y105 or aconservative substitution thereof, or Y106 or a conservativesubstitution thereof.
 25. The isolated ST2 antibody of claim 24, whereinsaid heavy chain variable region comprises S102 or a conservativesubstitution thereof, S103 or a conservative substitution thereof, D104or a conservative substitution thereof, and Y105 or a conservativesubstitution thereof.
 26. The isolated ST2 antibody of claim 25, whereinsaid heavy chain variable region comprises S102, S103, D104, and Y105.27. An isolated ST2 antibody comprising: a light chain variable domaincomprising: the LCDR1 sequence set forth in SEQ ID NO:107; the LCDR2sequence set forth in SEQ ID NO:118; and the LCDR3 sequence set forth inSEQ ID NO:129; and a heavy chain variable domain comprising: the HCDR1sequence set forth in SEQ ID NO:41; the HCDR2 sequence set forth in SEQID NO:52; and the HCDR3 sequence set forth in SEQ ID NO:63.
 28. Theisolated ST2 antibody of claim 27, wherein the light chain variabledomain comprises the amino acid sequence set forth in SEQ ID NO:96; andthe heavy chain variable domain comprises the amino acid sequence setforth in SEQ ID NO:30.
 29. The isolated ST2 antibody of claim 28,comprising a light chain and a heavy chain, wherein the light chaincomprises the amino acid sequence set forth in SEQ ID NO:85 and theheavy chain comprises the amino acid sequence set forth in SEQ ID NO:19.